Complex formation for parallel detection of multiple analytes in multiple applications

Abstract

The technology provided herein relates to multiplex methods and kits for detecting different analytes in a sample simultaneously, in particular by using premixed macromolecule complexes like complexes having a primary and a secondary antibody, wherein the primary antibodies contain identical identifier elements.

Description

[0001]RES-PA13-PCT COMPLEX^FORMATION^FOR^PARALLEL^DETECTION OF^MULTIPLE^ANALYTES IN^MULTIPLE^ APPLICATIONS FIELD^OF^THE^DISCLOSURE The^ technology^ provided^ herein^ relates^ to^multiplex^methods^ and^ kits^ for detecting^ different^ analytes^in^a^sample simultaneously,^in^particular^by^using^premixed^macromolecule^complexes^ like^ complexes^ having a^ primary and^ a^ secondary^ antibody,^ wherein^ the^ primary^ antibodies contain^identical^identifier^elements. BACKGROUND The^analysis^and^detection^of^small^quantities^of^analytes^like^RNA^or^proteins^in^biological^and^ non-biological^samples^has^become^a^routine^practice^in^the^clinical^and^analytical^environment.^ Numerous^analytical^methods^have^been^established^for^this^purpose.^Some^of^them^use^encoding^ techniques^assigning^a^particular^readable^code^to^a^specific^first^analyte^which^differs^from^a^code^ assigned^to^a^specific^second^analyte. One^of^the^prior^art^techniques^in^this^field^is^the^so-called^'single^molecule^fluorescence^in^situ^ hybridization'^(smFISH)^essentially^developed^to^detect^mRNA^molecules^in^a^sample.^In^Lubeck^ et^al.^(2014),^Single-cell^in^situ^RNA^profiling^by^sequential^hybridization,^Nat.^Methods 11(4),^p.^ 360-361,^ the^mRNAs^of^ interest^are^detected^via^specific^directly^ labeled^probe^sets.^After^one^ round^of^hybridization^and^detection,^the^set^of^mRNA^specific^probes^is^eluted^from^the^mRNAs^ and^the^same^set^of^probes^with^other^(or^the^same)^fluorescent^labels^is^used^in^the^next^round^ of^hybridization^and^imaging^to^generate^gene^specific^color-code^schemes^over^several^rounds.^ The^technology^needs^several^differently^tagged^probe^sets^per^transcript^and^needs^to^denature^ these^probe^sets^after^every^detection^round. A^further^development^of^this^technology^does^not^use^directly^labeled^probe^sets.^Instead,^the^ oligonucleotides^ of^ the^ probe^ sets^ provide^ nucleic^ acid^ sequences^ that^ serve^ as^ initiator^ for^ hybridization^chain^reactions^(HCR),^a^technology^that^enables^signal^amplification;^see^Shah^et^ al.^(2016),^In^situ^transcription^profiling^of^single^cells^reveals^spatial^organization^of^cells^in^the^ mouse^hippocampus,^Neuron^92(2),^p.^342-357. RES-PA13-PCT Immunohistochemistry^ (IHC)^ refers^ to^ a processes^of^detecting,^ localizing,^ and / or^quantifying^ antigens,^ such^ as^ a^ protein,^ in^ a^ biological^ sample^ using^ antibodies^ specific^ to^ the^ particular^ antigens.^IHC^provides^the^substantial^advantage^of^identifying^exactly^where^a^particular^protein^ is^located^within^the^tissue^sample.^It^is^also^an^effective^way^to^examine^the^tissues^themselves.^ In^situ^hybridization^(ISH)^refers^to^the^process^of^detecting,^localizing,^and^quantifying^nucleic^ acids.^Both^IHC^and^ISH^can^be^performed^on^various^biological^samples,^such^as^tissue^(e.g.^fresh^ frozen,^formalin^fixed,^paraffin^embedded)^and^cytological^samples.^Recognition^of^the^targets^can^ be^ detected^ using^ various^ labels^ (e.g.,^ chromogenic,^ fluorescent,^ luminescent,^ radiometric),^ irrespective^of^whether^the^target^is^a^nucleic^acid^or^an^antigen.^To^robustly^detect,^locate,^and^ quantify^ targets^ in^ a^ clinical^ setting,^ amplification^of^ the^ recognition^ event^ is^ desirable^ as^ the^ ability^to^confidently^detect^cellular^markers^of^low^abundance^becomes^increasingly^important^ for^diagnostic^purposes.^For^example,^depositing^at^the^marker's^site^hundreds^or^thousands^of^ label^molecules^in^response^to^a^single^antigen^detection^event^enhances,^through^amplification,^ the^ability^to^detect^that^recognition^event. However,^ conventional^ immunohistochemistry^ (IHC)^ methods^ do^ not^ necessarily^ provide^ adequate^signal^for^detection,^particularly^for^immunological^targets^that^are^weakly^expressed^or^ which^are^not^efficiently^targeted^by^existing^IHC^reagents. The^foregoing^immunohistochemical^ methods^for^target^analyte^labeling^can^e.g. be^limited^by^the^quantity^of^probe^dye^that^can^be^ delivered^to^specifically^label^each^of^the^target^analytes^in^the^sample.^Because^probe^dyes^are^ conjugated^to^specific^antibodies,^the^amount^of^probe^dye^that^can^be^applied^to^a^specific^location^ in^a^sample^depends^directly^on^the^number^of^probe^dye^moieties^that^are^directly^atached^to^an^ antibody^ that^ binds^ to^ an^ analyte^ at^ that^ location.^ Certain^ immunohistochemical^methods^ are^ therefore somewhat^ limited^ in^ their^ ability^ to^ amplify^ signals^ arising^ from^ particular^ target^ analytes^by^selectively^depositing^ larger^quantities^of^probe^dye^at^ locations^corresponding^to^ those^target^analytes. One^technique^based^on^Dako^ARKTMis^a^chromogenic^immunohistological^staining.^The^system^ is^designed^ for^ staining^with^mouse^primary^ antibodies^ on^ formalin-fixed,^ paraffin-embedded^ tissues,^cryostat^ tissues^or^cell^preparations^ from^any^species,^ including^mouse.^The^ technique^ used^in^this^system^are^based on^the^avidin-biotin^and^peroxidase^methodologies.^The^primary^ antibody^and^the^biotinylation^reagent^are^mixed^in^solution,^resulting^in^binding^of^biotinylated^ secondary^antibody^to^the^primary^antibody.^The^blocking^reagent,^containing^normal^serum,^is then^added^to^the^mixture.^The^immunoglobulin^present^in^the^blocking^reagent^binds^residual^ RES-PA13-PCT biotinylation^reagent^not^bound^to^the^primary^antibody.^The^biotin-labelled^primary^antibody^is^ then^ applied^ to^ the^ specimen.^ The^ specimen^ is^ next^ incubated^ with streptavidin-peroxidase,^ followed^by^reaction^with^diaminobenzidine / hydrogen^peroxide^as^substrate-chromogen.^ The^drawback^of^this^technique^are that^the^detection^system,^which^is^based^on^the^enzymatic^ reaction^of^diaminobenzidine / hydrogen^peroxide^as substrate-chromogen,^is^not^applicable^for^ parallel^protein^detection^(more^ than^2^proteins / ^detection).^Furthermore,^ ^ streptavidin^has^a^ strong^affinity^to^biotin,^forming^a^biotin-avidin^complex.^Increasing^number^of^parallel^protein^ detection^ can^ cause cross^ interaction^ among^ the^ different^ primary^ antibody^ and^ Fab-Biotin^ complex.^ Therefore, the^ methods^ for^ detection^ of^ multiple^ analytes^ known^ in^ the^ art^ have^ numerous^ disadvantages.^In^particular,^they^are^inflexible,^expensive,^complex,^time^consuming and^quite^ often^provide^non-accurate^results.^ Against^this^background,^it^is^an^object^underlying^the^present^disclosure^to^provide^a^method^by^ means^of^which^the^disadvantages^of^the^prior^art^methods^can^be^reduced^or^even^avoided. SUMMARY^OF^THE^DISCLOSURE The^ present^ disclosure^ pertains^ to^ novel^ multiplex^ methods^ and^ kits^ for^ detecting^ different^ analytes^in^a^sample^in^parallel^by^using^premixed^complexes.^In^particular,^the^present^disclosure relates^to^a^parallel^detection^of^analytes^by^primary^decodeable^complexes.^ In^a^first^aspect,^the^present^disclosure^pertains^to^methods for^detecting^different^analytes^in^a^ sample^simultaneously^comprising^the^steps^of:^ (A)^ contacting^ the^ sample^ with^ at^ least^ four^ (4)^ different^ sets^ of^ analyte-specific probes^ for^ detecting^of^at^ least^4^different^analytes,^each^set^ of^analyte-specific^probes^ interacting^with^a^ different^analyte,^wherein^each^analyte-specific^probe^is^a^complex^comprising (aa)^ a^ binding^ element^ (Y)^ that^ specifically^ interacts^ with^ one^ of the^ different^ analytes^ to^ be^ encoded,^wherein^each^binding^element^(Y)^of^each^set^of^analyte-specific^probes^comprises^an^ identical^identifier^element^(T),^and RES-PA13-PCT (bb)^an^identifier^element^(Z)^that^specifically^interact^and^bind^to^the^identifier^element^(T)^of^the^ binding^element^(Y),^wherein^the^identifier^element^(Z)^is^coupled^to^an^identifier^fluorophore^(f)^ or^an^identifier^oligonucleotide^(o)^that^is^used^to^identify^the^identity^of^the^identifier^element^ (Z),^wherein^ the^ identifier^ fluorophore^(f)^or^ the^ identifier^oligonucleotide^(o)^of^ the^ identifier^ element^(Z)^in^a^particular^set^of^analyte-specific^probes^differ^from^the^identifier^fluorophore^(f)^ or^the^identifier^oligonucleotide^(o)^of^the^identifier^element^(Z)^of^another^set^of^analyte-specific^ probes, wherein^ the^ binding^ element^ (Y)^ and^ the^ identifier^ element^ (Z)^ are^ premixed^ for^ each^ set^ of^ analyte-specific^ probes^ in^ a^ separate^ reaction^ tube^ to^ form^ complexes^ between^ the^ binding^ element^(Y)^and^the^identifier^element^(Z)^before^contacting^the^sample^with^the^different^sets^of^ analyte-specific^probes,^wherein^ the^ identifier^element^(Z)^ interacts^and^binds^ to^ the^ identical^ identifier^element^(T)^of^the^binding^element^(Y), (B)^detecting^the^signal^caused^by^the^identifier^fluorophore^(f)^coupled^to^the^identifier^element^ (Z);^or^ (C)^ i)^ contacting^ the^ sample^ with^ different^ set^ of^ signal^ oligonucleotides,^ each^ signal^ oligonucleotide^comprising: (aa)^a^ translator^connector^element^(C)^comprising^a^nucleotide^sequence^which^ is^essentially^ complementary^to^at^least^a^section^of^the^nucleotide^sequence^of^an^identifier^oligonucleotide^(o)^ comprised^in^an^identifier^element^(Z),^and (bb)^a^signal^element^and / or^an^additional^sequence^that^can^be^detected^by^hybridizing^to^one^or^ multiple^complementary^oligonucleotides^that^are^labeled^or ii)^contacting^the^sample^with^at^least^one^set^of^decoding^oligonucleotides^per^analyte,^wherein^ in^each^set^of^decoding^oligonucleotides^for^an^individual^analyte^each^decoding^oligonucleotide^ comprises: (aa)^an^ identifier^connector^element^(t)^comprising^a^nucleotide^sequence^which^ is^essentially^ complementary^to^at^least^a^section^of^the^identifier^oligonucleotide^(o)^of^an^identifier^element^ (Z)^of^the^corresponding^analyte-specific^probe^set,^and (bb)^a^translator^element^(c)^comprising^a^nucleotide^sequence^allowing^a^specific^hybridization^ of^a^signal^oligonucleotide; RES-PA13-PCT wherein^the^decoding^oligonucleotides^of^a^set^for^an^individual^analyte^differ^from^the^decoding^ oligonucleotides^ of^ another^ set^ for^ a^ different^ analyte^ in^ the^ first^ connect^ element^ (t);^ and^ contacting^the^sample^with^at^least^a^set^of^signal^oligonucleotides,^each^signal^oligonucleotide^ comprising: (aa)^a^ translator^connector^element^(C)^comprising^a^nucleotide^sequence^which^ is^essentially^ complementary^ to^ at^ least^ a^ section^ of^ the^ nucleotide^ sequence^ of^ a^ translator^ element^ (c)^ comprised^in^a^decoding^oligonucleotide,^and (bb)^a^signal^element^and / or^an^additional^sequence^that^can^be^detected^by^hybridizing^to^one^or^ multiple^complementary^oligonucleotides^that^are^labeled; (D) Detecting^ the^ signal^ caused^ by^ the^ signal^ element^ of^ section^ C)^ i)^ and / or^ C)^ ii);^ and^ optionally (E) Selectively^ removing^ of^ the^ signal^ oligonucleotides^ of^ section^ C)^ i)^ or^ the^ decoding^ oligonucleotides^ and^ signal^ oligonucleotides^ of^ of^ section^ C)^ ii)^ from^ the^ sample,^ thereby^ essentially^maintaining^the^specific^binding^of^the^analyte-specific^probes^to^the^analytes^to^be^ encoded;^and^optionally (F) Performing^ at^ least^ three^ (3)^ further^ cycles^ comprising^ steps^ C)^ to^ E)^ to^ generate^ an^ encoding^scheme^with^a^code^word^per^analyte,^wherein^in^particular^the^last^cycle^may^stop^with^ step^(D). In^ some^embodiments,^ the^present^disclosure^pertains^ to the^use^of^ complexes,^wherein^ each^ complex comprises^an antigen(analyte)-specific^primary^antibody^and^a^secondary^antibody^or^ an^antibody^fragment,^^wherein^the^secondary^antibody^or^the^antibody^fragment is^conjugated (tagged)^ with^ an oligonucleotide^ that^ is used^ to^ detect^ the^ antigens (analyts) of^ interest^ simultaneously.^The^complexes^are^formed^before^contacting^to^the^sample^in^multiple^reaction^ tubes^and^added^to^biological^specimens.^The^presence of^antigens (analytes) can^be^identified^via^ various^options^using^complementary^oligonucleotides.^This^allows^a^simultaneous detection^of^ analytes. In^a^second aspect,^embodiments^of^the^disclosure^pertains^to^a method^for^detecting^different^ analytes^ in^ a^ sample^ simultaneously,^ comprising:^ ^ contacting^ the^ sample^ to^ a^ population^ of^ separately^premixed^different^analyte-specific^probe^sets,^wherein^members^of^the^population^of^ each^analyte-specific^probe^set^comprises^complexes^having^ RES-PA13-PCT a)^a^binding^element^(Y)^that^specifically^interacts^with^one^of^the^different^analytes^to^be^encoded,^ wherein^each^binding^element^(Y)^of^each^set^of^analyte-specific^probes^comprises^an^identical^ identifier^element^(T),^and (b)^an^identifier^element^(Z)^that^specifically^interact^and^bind^to^the^identifier^element^(T)^of^the^ binding^element^(Y),^wherein^the^identifier^element^(Z)^is^coupled to an^identifier^oligonucleotide^ (o)^ that^ is^ used^ to^ identify^ the^ identity^ of^ the^ identifier^ element^ (Z),^ wherein^ the^ identifier^ oligonucleotide^(o)^of^the^identifier^element^(Z)^in^a^particular^set^of^analyte-specific^probes^differ^ from^ the^ identifier^ oligonucleotide^ (o)^ of^ the^ identifier^ element^ (Z)^ of^ another^ set^ of^ analyte- specific^probes,^ contacting^the^complexes^bound^to^the^analytes^to^a^first^decoder^element^population,^the^first^ decoder^element^population^comprising^probes^having^a^region^complementary^to^the^identifier^ oligonucleotide^(o)^and^a^region^having^a^first^signal-element^binding^domain;^^ contacting^ the^ first^ decoder^ element^ population^ bound^ to^ the^ complexes^ bound^ to^ the^ to^ the^ analytes^ with^ a^ first^ signal^ element^ probe^ comprising^ a^ first^ fluorophore;^ detecting^ a^ first^ fluorophore^emission^spectrum;^removing^the^first^decoder^element^population;^contacting^the^ complexes^bound^to^ the^analytes^ to^a^second^decoder^element^population,^ the^second^decoder^ element^ population^ comprising^ probes^ having^ a^ region^ complementary^ to^ the^ identifier^ oligonucleotide^ (o)^ element^ and^ a^ region^ having^ a^ second^ signal-element^ binding^ domain;^ contacting^ the^ identifier^ element^ bound^ analyte^ complex^ to^ a^ second^ signal^ element^ probe^ comprising^ a^ second^ fluorophore;^ detecting^ a^ second^ fluorophore^ emission^ spectrum;^ ^ and^ associating^ a^ pattern^ of^ first^ fluorophore^ emission^ spectra^ and^ second^ fluorophore^ emission^ spectra^ consistent^with^ an^order^of^ addition^of^ first^decoder^ element^populations^ and^ second^ decoder^element^populations. In^a^second aspect,^embodiments^of^the^disclosure^pertains^to^a method^for^detecting^different^ analytes^in^a^sample^simultaneously^comprising:^a)^contacting^a^biological^sample^with^at^least^ four^(4)^different^sets^of^analyte-specific^antibodies^or^antibody^fragments^for^detecting^at^least^ four^ (4)^ different^ analytes,^wherein^ the^ antibodies^ or^ antibody^ fragments^ of^ each^ set^ bind^ to^ different^analytes^compared^to^the^antibodies^or^antibody^fragments^of^another^set,^wherein^the^ RES-PA13-PCT antibodies^or^antibody^fragments^in^all^sets^comprise^the^same^(Fc)^region,^wherein^the^different^ antibodies^ or^ antibody^ fragments^ in^ the^ different^ sets^ are^ separately^ premixed^ in^ each^ set^ of^ analyte-specific^probes^with^secondary^antibodies^or^antibody^fragments^that^binds^to^the^(Fc)^ region^of^the^analyte-specific^antibodies^or^antibody^fragments^to^form^complexes^between^the^ analyte-specific^ antibodies^ or^ antibody^ fragments^ and^ the^ secondary^ antibodies,^ wherein^ the^ secondary^ antibodies^ comprise^ each^ an^ identifier^ oligonucleotide^ (o),^ wherein^ the^ identifier^ oligonucleotide^(o)^^comprised^on^the^secondary^antibodies^in^each^set^differs^from^the^identifier^ oligonucleotide^ (o)^ in^ another^ set;^ b)^ contacting^ said^ complexes^ bound^ to^ the^ analytes^ with^ decoding^oligonucleotides;^c)^contacting^said^decoding^oligonucleotides^bound^to^said^complexes^ with^signal^oligonucleotides;^d)^detection^of^the^signal^oligonucleotides^bound^to^said^decoding^ oligonucleotides^comprised^in^said^complexes^bound^to^the^analytes. In^ a^ third^ aspect,^ embodiments^ of^ the^ disclosure^ pertains^ to^ a method^ for^ detecting^ different^ analytes^in^a^sample^simultaneously^comprising^the^steps^of:^ (A)^ contacting^ the^ sample^ with^ at^ least^ four^ (4)^ different^ sets^ of^ analyte-specific^ probes^ for^ detecting^of^at least^4^different^analytes,^each^set^ of^analyte-specific^probes^ interacting^with^a^ different^analyte,^wherein^each^analyte-specific^probe^is^a^complex^having (aa)^ a^ binding^ element^ (Y)^ that^ specifically^ interacts^ with^ one^ of^ the^ different^ analytes^ to^ be^ encoded,^wherein^each^binding^element^(Y)^of^each^set^of^analyte-specific^probes^comprises^an^ identical^identifier^element^(T),^and (bb)^an^identifier^element^(Z)^that^specifically^interacts and^binds to^the^identifier^element^(T)^of^ the^binding^element^(Y),^wherein^the^identifier^element^(Z)^is^coupled^to^an^identifier^fluorophore^ (f)^and / or^an^identifier^oligonucleotide^(o)^that^is^used^to^identify^the^identity^of^the^identifier^ element^ (Z),^wherein^ the^ identifier^ fluorophore^ (f)^ or^ the^ identifier^ oligonucleotide^ (o)^ of^ the^ identifier^ element^ (Z)^ in^ a^ particular^ set^ of^ analyte-specific^ probes^ differ^ from^ the^ identifier^ fluorophore^(f)^or^the^identifier^oligonucleotide^(o)^of^the^identifier^element^(Z)^of^another^set^of^ analyte-specific^probes, wherein^the^binding^element^(Y)^and^the^identifier^element^(Z)^are^premixed^in^each^set^of^analyte- specific^probes^to^form^complexes^between^the^binding^element^(Y)^and^the^identifier^element^(Z)^ RES-PA13-PCT before^ contacting^ the^ sample^ with^ the^ different^ sets^ of^ analyte-specific^ probes,^ wherein^ the^ identifier^element^(Z)^interacts^and^binds^to^the^identifier^element^(T)^of^the^binding^element^(Y), (B)^detecting^the^signal^caused^by^the^identifier^fluorophore^(f)^coupled^to^the^identifier^element^ (Z);^or^ (C)^ i)^ contacting^ the^ sample^ with^ different^ set^ of^ signal^ oligonucleotides,^ each^ signal^ oligonucleotide^comprising: (aa)^a^ translator^connector^element^(C)^comprising^a^nucleotide^sequence^which^ is^essentially^ complementary^to^at^least^a^section^of^the^nucleotide^sequence^of^an identifier^oligonucleotide^(o)^ comprised^in^an^identifier^element^(Z),^and (bb)^a^signal^element^and / or^an^additional^sequence^that^can^be^detected^by^hybridizing^to^one^or^ multiple^complementary^oligonucleotides^that^are^labeled^or ii)^contacting^the^sample^with^at^least^one^set^of^decoding^oligonucleotides^per^analyte,^wherein^ in^each^set^of^decoding^oligonucleotides^for^an^individual^analyte^each^decoding^oligonucleotide^ comprises: (aa)^an^ identifier^connector^element^(t)^comprising^a^nucleotide^sequence^which^ is^essentially^ complementary^to^at^least^a^section^of^the^identifier^oligonucleotide^(o)^of^an^identifier^element^ (Z)^of^the^corresponding^analyte-specific^probe^set,^and (bb)^a^translator^element^(c)^comprising^a^nucleotide^sequence^allowing^a^specific^hybridization^ of^a^signal^oligonucleotide; wherein^the^decoding^oligonucleotides^of^a^set^for^an^individual^analyte^differ^from^the^decoding^ oligonucleotides^ of^ another^ set^ for^ a^ different^ analyte^ in^ the^ first^ connect^ element^ (t);^ and^ contacting^the sample^with^at^least^a^set^of^signal^oligonucleotides,^each^signal^oligonucleotide^ comprising: (aa)^a^ translator^connector^element^(C)^comprising^a^nucleotide^sequence^which^ is^essentially^ complementary^ to^ at^ least^ a^ section^ of^ the^ nucleotide^ sequence^ of^ a^ translator^ element^ (c)^ comprised^in^a^decoding^oligonucleotide,^and (bb)^a^signal^element^and / or^an^additional^sequence^that^can^be^detected^by^hybridizing^to^one^or^ multiple^complementary^oligonucleotides^that^are^labeled; (D) Detecting^ the^ signal^ caused^ by^ the^ signal^ element^ of^ section^ C)^ i)^ and / or^ C)^ ii);^ and^ optionally; RES-PA13-PCT (E) Selectively^ removing^ the^ decoding^ oligonucleotides^ of^ and^ signal^ oligonucleotides^ of^ section^ii)^from^the^sample,^thereby^essentially^maintaining^the^specific^binding^of^the^analyte- specific^probes^to^the^analytes^to^be^encoded;^and^optionally (F) Performing^ at^ least^ three^ (3)^ further^ cycles^ comprising^ steps^ C)^ ii)^ to^ E)^ to^ optionally^ generate^an^encoding^scheme^with^a^code^word^per^analyte,^wherein^in^particular^the^last^cycle^ may stop^with^step^(D). In^ a^ third^ aspect,^ embodiments^of^ the^disclosure^ pertains^ to^ a^mixture^ for^detecting^different^ analytes^in^a^sample^simultaneously,^wherein^the^mixture^comprises^at^least^four^(4)^different^sets^ of^analyte-specific^probes^for^encoding of^at^least^4^different^analytes,^each^set^of^analyte-specific^ probes^ interacting^ with^ a^ different^ analyte,^ wherein^ each^ analyte-specific^ probe^ comprises^ complexes^having (aa)^ a^ binding^ element^ (Y)^ that^ specifically^ interacts^ with^ one^ of^ the^ different^ analytes^ to^ be^ encoded,^wherein^each^binding^element^(Y)^of^each^set^of^analyte-specific^probes^comprises^an^ identical^identifier^element^(T),^and (bb)^an^identifier^element^(Z)^that^specifically^interact^and^bind^to^the^identifier^element^(T)^of^the^ binding^element^(Y),^wherein^the^identifier^element^(Z)^is^coupled^to^an^identifier^fluorophore^(f)^ or^an^identifier^oligonucleotide^(o)^that^is^used^to^identify^the^identity^of^the^identifier^element^ (Z),^wherein^ the^ identifier^ fluorophore^(f)^or^ the^ identifier oligonucleotide^(o)^of^ the^ identifier^ element^(Z)^in^a^particular^set^of^analyte-specific^probes^differ^from^the^identifier^fluorophore^(f)^ or^the^identifier^oligonucleotide^(o)^of^the^identifier^element^(Z)^of^another^set^of^analyte-specific^ probes. In^particular,^the^present^description describes^the^combinatorial^use^of^antibodies^together^with^ secondary^antibodies,^FABs^or^proteins^that^specially^bind^to^an^antibody^or^protein^that^recognize^ and^bind^specially^another^protein. Therefore,^the^present^disclosure relates^to^a^parallel^detection^ of^analytes^by^primary^decodeable^complexes.^ This^refers^to^complexes^of^an^antigen-specific^of^ primary^antibody^and^am^secondary^antibody^or^Fab^conjugated^oligonucleotide^that^are^used^to^ detect^antigens^of^interest^simultaneously.^The^complexes^between^the^first^antibodies^and^the^ secondary^bantibodies^are^formed^in^multiple^reaction^tubes^and^added^to^biological^specimens.^ RES-PA13-PCT The^ present^ of^ antigens^ can^ be^ identified^ via^ various^ options^ using^ complementary^ oligonucleotides.^This^allows^a^simultaneous^detection^of^analytes,^in^particular^of^proteins. In^a^further aspect,^embodiments^of^the^disclosure^pertains^to^a^kit^for^detecting^different^analytes^ in^a^sample^simultaneously,^wherein^the^kit^comprises^separately^at^least^four^(4)^different^sets^of^ analyte-specific^probes^for^encoding^of^at^ least^4^different^analytes,^each^set^of^analyte-specific^ probes^ interacting^ with^ a^ different^ analyte,^ wherein^ each^ analyte-specific^ probe^ comprises^ complexes^having (aa)^ a^ binding^ element^ (Y)^ that^ specifically^ interacts^ with^ one^ of^ the^ different^ analytes^ to^ be^ encoded,^wherein^each^binding^element^(Y)^of^each^set^of^analyte-specific^probes^comprises^an^ identical^identifier^element^(T),^and (bb)^an^identifier^element^(Z)^that^specifically^interact^and^bind^to^the^identifier^element^(T)^of^the^ binding^element^(Y),^wherein^the^identifier^element^(Z)^is^coupled^to^an^identifier^fluorophore^(f)^ or^an^identifier^oligonucleotide^(o)^that^is^used^to^identify^the identity^of^the^identifier^element^ (Z),^wherein^ the^ identifier^ fluorophore^(f)^or^ the^ identifier^oligonucleotide^(o)^of^ the^ identifier^ element^(Z)^in^a^particular^set^of^analyte-specific^probes^differ^from^the^identifier^fluorophore^(f)^ or^the^identifier^oligonucleotide^(o)^of^the^identifier^element^(Z)^of^another^set^of^analyte-specific^ probes. Before^the^disclosure^is^described^in^detail,^it^is^to^be^understood^that^this^disclosure^is^not^limited^ to^the^particular^component^parts^of^the^steps^of^the^methods^described.^It^is^also^to^be^understood^ that^the^terminology^used^herein^is^for^purposes^of^describing^particular^embodiments^only,^and^ is^not^intended^to^be^limiting.^It^must^be^noted^that,^as^used^in^the^specification^and^the^appended^ claims,^the^singular^forms^"a,"^"an"^and^"the"^include^singular^and / or^plural^referents^unless^the^ context^clearly^dictates^otherwise.^It^is^moreover^to^be^understood^that,^in^case^parameter^ranges^ are^ given^ which^ are^ delimited^ by^ numeric values,^ the^ ranges^ are^ deemed to^ include^ these^ limitation^values.^ BRIEF^DESCRIPTION^OF^THE^DRAWINGS Fig.^1^ shows^ examples^ of^ 1)^ identifier^ element^ (Z)^ that^ specifically^ interact^ and^ bind^ to^ the^ identifier^element^(T)^of^a binding^element^(Y)^and^examples^2) of^^complexes^comprising^ RES-PA13-PCT a^binding^element^(Y)^that^specifically^interacts^with^one^of^the^different^analytes^to^be^ encoded,^ wherein^ each^ binding^ element^ (Y)^ of^ each^ set^ of^ analyte-specific^ probes^ comprises^ an^ identical^ identifier^ element^ (T),^ and (bb)^ an^ identifier^ element^ (Z)^ that^ specifically^ interact^ and^ bind^ to^ the^ identifier^ element^ (T)^ of^ the^ binding^ element^ (Y),^ wherein^ the^ identifier^ element^ (Z)^ is^ coupled^ to^ an^ identifier^ fluorophore^ (f)^ or^ an^ identifier^oligonucleotide^(o)^that^is^used^to^identify^the^identity^of^the^identifier^element^ (Z),^ wherein^ the^ identifier^ fluorophore^ (f)^ or^ the^ identifier^ oligonucleotide^ (o)^ of^ the^ identifier^element^(Z)^in^a^particular^set^of^analyte-specific^probes^differ^from^the^identifier^ fluorophore^(f)^or^the^identifier^oligonucleotide^(o)^of^the^identifier^element^(Z)^of^another^ set^of^analyte-specific^probes. Fig.^2 are^schematic^diagrams^showing^methods^for^sample^analysis according^to^the^present^ disclosure. Fig.^3^ shows^a^cryosection^of^murine^kidney^was^probed^ for^nestin.^Rabbit^primary^antibody^ against^nestin^was^prelabelled^with^Alexa^Flour^647^conjugated^ to^goat^anti-rabbit^ IgG^ (upper^panel)^or^goat^anti^rabbit^Fab^(lower^panel)^with^a^ratio^between^primary^antibody^ and^ secondary^ antibody^ of^ ^ 1:0.2^ and^ 1:2^ (molecules / molecules),^ respectively.^ The^ prelabelling^was^performed^for^30^minutes^and^residual^secondary^antibody^or^Fab^not^ bound^to^primary^antibody^was^blocked^with^0.1%^v / v^normal^rabbit^serum^for^5^min.^The^ antibody^complexes^were^added^to^tissues^and^incubated^overnight.^Images^were^captured^ with^ ZEISS^ Celldiscoverer^ 7^ using^ a^ Apochromat^ 5X / 0.35^ objective^ and^ 2X^ Tubelens.^ (Exposure^time:^1^second^and^0.1^second^for^Alexa^Flour^647^and^DAPI,^respectively). Fig.^4 shows^a^cyrosection^of^murine^kidney^probed^for^Nestin.^Detection^of^Nestin was^done^by^ precomplexed^ primary^ antibody^ (Abcam^ ab176571)^with^ oligos^ conjugated^ secondary^ antibody^ followed^ by^ complementary^ oligos^ conjugated^ to^ fluorophores^ (A).^ The^ localization^of^the^primary-secondary^antibody^complexes^was^confirmed^by^probing^the tissue^ with^ fluorophores^ conjugated^ antibody^ complemented^ to^ the^ oligo^ conjugated^ secondary^antibody^(B).^^^ ^^^^^^^^^ DETAILED^DESCRIPTION^OF^THE DISCLOSURE RES-PA13-PCT The^present disclosure^features^methods^for^performing^multiplexed^labeling,^identification and^ quantification^ of^ target^ analytes^ in^ a^ biological^ sample.^ The^methods^ can^ be^ used^ to^ perform^ multiple^cycles^of^target^analyte^labeling,^detection,^and^removal^of^certain^agents^involved^in^the^ labeling^process,^without^disrupting^antibody-analyte^binding^in^the^sample.^Instead,^removal^of^ agents^involved^in^the^labeling^process^is^performed^by^dehybridizing^the^agents^under^relatively^ mild^conditions,^preserving^sample^integrity^and^ensuring^that^removal^of^the^agents^during^each^ labeling^ and^detection^ cycle^ is^ nearly^ complete.^ As^ a^ result,^ little^ or^ no^ cross-species^ labeling^ occurs.^ Instead,^ each^ target^ analyte^ in^ the^ sample^ can^ be^ selectively^ labeled^ with^ a^ different^ labeling^species,^and^deposition^of^each^labeling^species^is^highly^constrained^to^regions^of^the^ sample^that^specifically^correspond^to^the^location^of^a^specific^target^analyte. With^ the methods^ of^ the^ present^ disclosure^ multiplexed^ labeling,^ identification,^ signal^ amplification,^and^quantification^of^target^analytes^in^a^biological^sample may^be^performed.^The^ methods^ of^ the^ present^ disclosure^ can^ be^ used^ to^ perform^ multiple^ cycles^ of^ target^ analyte^ labeling,^ detection,^ and^ removal^ of^ certain^ agents^ involved^ in^ the^ labeling^ process,^ without^ disrupting^ antibody-analyte^binding^ in^ the^ sample.^ Instead,^ removal^ of^ agents^ involved^ in^ the^ labeling^ process^ is^ performed^ by^ dehybridizing^ the^ agents^ under^ relatively^ mild^ conditions,^ preserving^sample^ integrity^and^ensuring^ that^removal^of^ the^ agents^during^each^ labeling^and^ detection^cycle^is^nearly^complete.^As^a^result,^little^or^no^cross-species^labeling^occurs.^Instead,^ each^target^analyte^in^the^sample^can^be^selectively^labeled^with^a^different^labeling^species,^and^ deposition^of^each^labeling^species^is^highly^constrained^to^regions^of^the^sample^that^specifically^ correspond^to^the^location^of^a^specific^target^analyte.The^present^disclosure^describes^the^usage^ of^a^set^of^labeled^and^unlabeled^nucleic^acid^sequences^for^specific^quantitative^and / or^spatial^ detection^of^different^analytes^ in^parallel^via^specific^hybridization.^The^ technology^ allows^ the^ discrimination^ of^ more^ different^ analytes^ than^ different^ detection^ signals^ are^ available.^ The^ discrimination^may^be^realized^via^sequential^signal-coding^of^the^analytes^achieved^by^several^ cycles of^specific^hybridization,^detection^of^signals^and^selective^elution^of^the^hybridized^nucleic^ acid^sequences. In^contrast^to^other^state-of-the-art^methods,^the^oligonucleotides^providing^the^detectable^signal^ are^not^directly^interacting^with^sample-specific^analyte-specific^probes^but^may^be^alsomediated^ by^so^called^“decoding-oligonucleotides”.^This^mechanism^decouples^the^dependency^between^the^ analyte-specific^ probes and^ the^ signal^ oligonucleotides.^ The^ use^ of^ decoding-oligonucleotides^ allows^ a^much^ higher^ flexibility^while^ dramatically^ decreasing^ the^ number^ of^ different^ signal^ RES-PA13-PCT oligonucleotides^ needed^which^ in^ turn^ increases^ the^ coding^ capacity^ achieved^with^ a^ certain^ number^of^detection^rounds. The^utilization^of^decoding-oligonucleotides^leads^to a^sequential^signal-coding^technology^that^is^ more^flexible,^cheaper,^simpler,^faster^and / or^more^accurate^than^other^methods. In^particular,^the^step^of^premixing^the^binding^elements (Y) with^the identifier^elements (Z) in^ multiple^ reaction^ tubes (for^ each^ type^ of^ analyte-specific^ probes one^ separate^ tube)^ to^ form^ complexes^before^contacting^the^sample^with^these^complexes^has^the^advantage^that: 1) Reduction^of^experimental^complexity^I:^ Usually^primary^ABs^generated^in^different^species^(goat,^rabbit,^chicken^etc.)^must^be^used^ for^multiplex^protein^detection^(see^table^1a).^If^a^complex^between^primary^antibody^ (primary^AB) and^secondary^antibody^(secondary^AB)^or secondary^antibody^ FAB fragment is^formed^before^applying^primary^AB^to^the^sample, different^primary^ABs^generated^in^the^ same^species^can^be^used^(see^table^1b^below)^^^^^ Table^1a: Table^1b: RES-PA13-PCT Anti^T2^ specific^for^ T2 Human AB rabbit rabbit Fluorophore^2 goat Anti^T3^ specific^for^ T3 Human AB rabbit rabbit Fluorophore^3 goat 2) Reduction^of^experimental^complexity^II:^ If^the^the detection^is^achieved^by^an^oligonucleotide^coupled^to^the^AB,^complex^formation^ prior^binding^to^the^sample^may^be^of^advantage.^In^this^case,^a^secondary^AB^(or^FAB)^can^be^ used^ for^ the^ coupling^ with^ different^ oligonucleotides.^ The^ advantage^ here^ is^ that^ not^ all^ primary^ABs^must^be^linked^to^oligonucleotides^but^only^a^limited^library^of^secondary^ABs.^ Secondary^ABs^(or^FABs)^could^be^linked^to^oligos^in^an^much^greater^set^up^than^individual^ primary^ABs.^ 3) Reduction^of^cost^and^time: Labeling^of^primary ABs^is^very^expensive^an^time^consuming^because^the^scale^is^small.^On^ the^other^side,^a^labeling^of^secondary^ABs^(or^FAB)^can^be^done^at^a^larger^scale^and^more^ cost^effective.^The^labelled^secondary^AB^(orFAB)^can^be^used^in^a^versatile^and^ubiquitous^ way^which^is^not^possible^with^fluorophore^labelled^primary^ABs.^A^complex^pre-formation^ of^primary^AB^and^fluorophore^labelled^secondary^AB^(orFAB)^^can^substitute^for^fluorophore^ labelled^primary^AB. 4) Increasing^sensitivity: In^ highly^ multiplexed^ protein detection^ systems,^ labeled^ primary^ ABs^ are^ used^ to^ avoid^ problems^in^compatibility^with^secondary^ABs.^For^these^assays,^the^use^of^labeled^primary^ ABs^limits^the^sensitivity^by^the^number^of^labels^coupled^to^the^primary^ABs.^If^complex^pre- formation^between^primary^ABs^and^labeled^secondary^ABs^can^be^performed,^the^sensitivity^ can^ increase^because^multiple^secondary^ABs^can^bind^to^ the^primary^AB^and^each^of^ the^ secondary^ABs^are^labeled^multiple^times. 5) Reduction^of^time: Preformation^of^a^complex^between^primary^AB^and^labelled,^secondary^AB^(or^FAB)^can^be^ done^ in^ an^ highly^ parallel^ way.^ After^ complex^ pre-formation,^ multiple^ complexes^ can^ be^ RES-PA13-PCT incubated^ with^ the^ sample^ simultaneously.^ Thereafter,^ a^ sequential^ detection^ can be^ performed. 6) Reducing^background^effects^I: Background^ effects^ can^ occur^ by^ primary^ and^ secondary^ ABs.^ The^ background^ effect^ by^ secondary^ABs^can^be^minimized^by^complex^pre-formation^between^primary^and^secondary^ AB^if^the^concentration^of^the secondary^AB^is^lower^than^the^concentration^oft^he^primary^ AB.^In^this^case,^the^sencondary^AB^is^almost^completely^bound^to^the^primary^AB^and^no^ secondary^AB^is^left^over. A.^Definitions According^to^the^present^disclosure^an^"analyte"^is^the^subject^to^be^specifically^detected^as^being^ present^or^absent^in^a^sample^and,^in^case^of^its^presence,^to^encode^it.^It^can^be^any^kind of^entity,^ including^a^protein,^polypeptide,^protein^or^a^nucleic^acid^molecule^(e.g.^RNA,^PNA^or^DNA)^of^ interest.^The^analyte^provides^at^least^one^site^for^specific^binding^with^analyte-specific^probes.^ Sometimes^ herein^ the^ term^ "analyte"^ is^ replaced^ by^ "target".^ An^ "analyte"^ according^ to^ the^ disclosure^ incudes^ a^ complex^ of^ subjects,^ e.g.^ at^ least^ two^ individual^ nucleic^ acid,^ protein^ or^ peptides^molecules.^In^an^embodiment^of^the^disclosure an^"analyte"^excludes^a^chromosome.^In^ another^ embodiment^ of^ the^ disclosure an^ "analyte"^ excludes^ DNA. In^ an^ advantageous^ embodiment,^the^analyte^is^a^protein / polypeptide. As^used^herein,^the^terms“contacts”^and“contacting”^mean^that^an^agent,^species,^moiety,^or^other^ element^is^brought^into^association^with^a^sample,^or^another^agent,^species,^moiety,^or^element,^ such^ that^ the^ two^ interact^ with^ one^ another.^ For^ example,^ when^ the^ sample^ ^ comprising^ the^ analytes^ is “contacted”^ with^ the^ different^ sets^ of^ analyte-specific^ probes,^ the^ analyte-specific^ probes^ are^brought^ into^ close^ enough^association^with^ the^ sample^ that^ they^ interact^with^ the^ anayltes^in^the^sample.^^ In^some^embodiments,^an^analyte^may^be^a^"coding^sequence", “encoding^sequence”,^"structural^ nucleotide^sequence"^or^"structural^nucleic^acid^molecule"^which^refers^to^a^nucleotide^sequence^ that^ is^ translated^ into^ a^ polypeptide,^ usually^ via^ mRNA,^ when^ placed^ under^ the^ control^ of^ appropriate^regulatory^sequences.^The^boundaries^of^the^coding^sequence^are^determined^by^a^ translation^start^codon^at^the^5'-terminus^and^a^translation^stop^codon^at^the^3'-terminus.^A^coding^ RES-PA13-PCT sequence^can^include,^but^is^not^limited^to,^genomic^DNA,^cDNA,^EST^and^recombinant^nucleotide^ sequences. A^"sample"^as^referred^to^herein^is^a^composition^in^liquid^or^solid^form^suspected^of^comprising^ the^analytes^to^be^encoded. In^particular,^the^sample^is^a^biological^sample,^preferably^comprising^ biological^ tissue,^ further^preferably^ comprising^biological^ cells^ and / or^ extracts^ and / or^part^ of^ cells.^For example,^the^cell^is^a^prokaryotic^cell^or^a^eukaryotic^cell,^in^particular^a^mammalian^cell,^ in^particular^a^human^cell.^ In^some^embodiments,^the^biological^tissue,^biological^cells,^extracts^ and / or^part^of^cells^are^fixed.^In^particular,^the^analytes^are^fixed^in^a^permeabilized^sample,^such^ as^a^cell-containing^sample. As^used^in^the^present^disclosure,^"cell",^"cell^line",^and^"cell^culture"^can^be^used^interchangeably^ and^all^such^designations include^progeny.^Thus,^the^words^"transformants"^or^"transformed^cells"^ include^the^primary^subject^cell^and^cultures^derived^therefrom^without^regard^for^the^number^of^ transfers.^It^is^also^understood^that^all^progeny^may^not^be^precisely^identical^in^DNA^content,^due^ to^ deliberate^ or^ inadvertent^ mutations.^ Mutant^ progeny^ that^ have^ the^ same^ functionality^ as^ screened^for^in^the^originally^transformed^cell^are^included.^ An^“encoding^scheme”^may^describe^a^set^of^code^words^that^are^associated^with^the^analytes^to^ be^detected.^Each^code^word^refers^to^one^of^the^analytes^and^can^be^distinguished^from^all^other^ code^words.^A^code^word^hereby^is^a^sequence^of^signs^provided^by^the^detection^cycles^of^the^ method.^A^sign^within^a^code^word^is^a^detectable^signal^or^the^absence^of^a^signal.^A^code^word^ does^not^need^to^comprise^of^all^different^signals^used^in^the^method.^The^number^of^signs^in^a^ code^word^is^defined^by^the^number^of^detection^cycles. An^"oligonucleotide"^as^used^herein,^refers^to^s^short^nucleic^acid^molecule,^such^as^DNA,^PNA,^ LNA^ or^ RNA.^ The^ length^ of^ the^ oligonucleotides^ is^ within^ the^ range^ 4-200^ nucleotides^ (nt),^ preferably^6-80^nt,^more^preferably^8-60^nt,^more^preferably^10-50^nt,^more^preferably^12^to^35^ depending^on^the^number^of^consecutive^sequence^elements.^The^nucleic^acid^molecule^can^be^ fully^or^partially^single-stranded.^The^oligonucleotides may^be^linear^or^may^comprise^hairpin^or^ loop^ structures.^ The^ oligonucleotides^ may^ comprise^ modifications^ such^ as^ biotin,^ labeling^ moieties,^blocking^moieties,^or^other^modifications. RES-PA13-PCT The^"analyte-specific^probe"^is^a^complex^consisting of^at^least^two^elements,^namely^the^so-called^ binding^element^(Y)^which^specifically^interacts^with^one^of^the^analytes,^wherein^each^binding^ element^(Y)^of^each^set^of^analyte-specific^probes^comprises^an^identical^identifier^element^(T),^ and^a^so-called^identifier^element^(Z)^that^specifically^interacts^and^bind^to^the^identical^identifier^ element^(T)^of^the^binding^element^(Y)^and^comprises an^identifier^fluorophore^(f)^or^an^identifier^ oligonucleotide^ (o).^ The^ binding^ element^ (Y)^ may^ be^ a^ nucleic^ acid^ such^ as^ a^ hybridization^ sequence^or^an^aptamer,^or^a^peptidic^structure^such^as^an^antibody.^ In^ particular,^ the^ binding^ element^ (Y)^ is^ an^ antibody,^ an^ antibody^ fragment,^ an^ aptamer^ or^ a^ nucleic^acid^that^specifically^interacts^and^binds^to^the^analyte, wherein^in^particular^the^antibody^ fragment^is^selected^from^the^group^consisting^of^a^Fab,^scFv,^single^domain,^bis^scFv,^Fab2,^Fab3,^ minibody,^diabody,^triplebody,^tetrabody^and^tandab. In^particular,^ the^ identifier^element^(Z)^ is^an^antibody,^an^antibody^ fragment,^an^aptamer^or^ a^ nucleic^acid^that^specifically^interacts^and^binds^to^the^analyte, wherein^in^particular^the^antibody^ fragment^is^selected^from^the^group^consisting^of^a^Fab,^scFv,^single^domain,^bis^scFv,^Fab2,^Fab3,^ minibody,^diabody,^triplebody,^tetrabody^and^tandab. In^an^advantageous^embodiment^of^the^present^disclosure,^the^binding^element^(Y)^is^an^antibody or an^ antibody^ fragment like^ a^ Fab,^ scFv,^ single^domain,^ bis^ scFv,^ Fab2,^ Fab3 fragment^ and^ the^ identifier^element^(Z)^is also^a^an^antibody or an^antibody^fragment like^a^Fab,^scFv,^single^domain,^ bis^scFv,^Fab2,^Fab3fragment.^In^an^advantageous^embodiment^of^the^present^disclosure,^the^binding^ element^(Y)^is^an^antibody and^the^identifier^element^(Z)^is^a secondary antibody. In^an^ advantageous^ embodiment^of^ the^present^disclosure,^ the^ analyte^ is^ a^protein / polypeptide comprised^in^a^biological^sample,^the^binding^element^(Y)^is^an^antibody^or^an^antibody^fragment^ comprising^a^(Fc)^region^and^the^identifier^element^(Z)^is^a^secondary^antibody,^a^Fab,^Fab2 and / or^ a^Fab3^fragment.^ In^particular,^in^some^embodiments^the^binding^element^(Y)^comprises^moieties^which^are^affinity^ moieties^from^affinity^substances^or^affinity^substances^in^their^entirety^selected^from^the^group^ consisting^ of^ antibodies,^ antibody^ fragments,^ receptor^ ligands,^ enzyme^ substrates,^ lectins,^ cytokines,^ lymphokines,^ interleukins,^ angiogenic^ or^ virulence^ factors,^ allergens,^ peptidic^ RES-PA13-PCT allergens,^ recombinant^ allergens,^ allergen-idiotypical^ antibodies,^ autoimmune-provoking^ structures,^ tissue-rejection-inducing^ structures,^ immunoglobulin^ constant^ regions^ and^ their^ derivatives,^ mutants^ or^ combinations^ thereof.^ In^ further^ advantageous^ embodiments,^ the^ antibody^fragment^is^a^Fab,^an^scFv;^a^single^domain,^or^a^fragment^thereof,^a^bis^scFv,^Fab2,^Fab3,^ minibody,^maxibody,^diabody,^triabody,^tetrabody^or^tandab,^in^particular^a^single-chain^variable^ fragment^(scFv). The^"identical^identifier^element^(T)"^as^comprised^in^a^binding^element^(Y)^of^an analyte-specific^ probe is^ identical in^ its^ sequence^ compared^ to^other^ identical^ identifier^ elements (T) in^other^ binding^ element^ (Y)^ of another analyte-specific^probe.^ In^other^words,^ the^ identical identifier^ elements (T) comprised^ in^ all^ binding^ elements^ (Y)^ in^ all^ sets^ of^ analyte-specific^ probes^ are^ identical. Or,^ in^other^words,^ there^ is^only^one^ identical identifier^element^(T)^sequence^ for^all^ binding^ elements^ (Y)^ used^ in^ a^ approach.^ Am^ identical^ identifier^ element^may^ be^ a^ sequence^ element^of^the^binding^element^(Y),^attached^directly^or^by^a^linker,^a^covalent^bond^or^high^affinity^ binding^modes,^e.g.^antibody-antigen^interaction,^streptavidin-biotin^interaction^etc.^ It^ is^understood^that^the^term^"analyte^specific^probe"^includes^a^plurality^of^complexes which^ may^differ^in^their^binding^elements^(Y)^in^a^way^that^each^probe^binds^to^the^same^analyte^but^ possibly^ to^ different^ parts^ thereof,^ for^ instance^ to^ different^ (e.g.^ neighboring)^ or^ overlapping^ sections^of^the^polypeptide^sequence^comprised^by^the^protein to^be^detected.^However,^each^of^ the^plurality^of^the^probes^comprises^the^same^identical identifier^element^(T). A^ "decoding^ oligonucleotide"^ or^ an^ “adapter”^ or^ a^ / adapter^ segment”^ consists^ of^ at^ least^ two^ sequence^ elements.^ One^ sequence^ element^ that^ can^ specifically^ bind^ to^ a^ unique^ identifier^ sequence,^referred^to^as an^“identifier^connector^element^“(t)^or "first^connector^element"^(t),^and^ a^ second^ sequence^ element^ specifically^ binding^ to^ a^ signal^ oligonucleotide,^ referred^ to^ as^ "translator^ element"^ (c).^ The^ length^ of^ the^ sequence^ elements^ is^ within^ the^ range^ 8-60^ nt,^ preferably^ 12-40^ nt,^ more^ preferably^ 14-20^ nt,^ de-pending^ on^ the^ number^ of^ analytes^ to^ be^ encoded^ in^ parallel,^ the^ stability^ of^ interaction^ needed^ and^ the^ number^ of^ different^ signal^ oligonucleotides^used.^The^length^of^the^two^sequence^elements^may^or^may^not^be^the^same. In^some^advantageous embodiments,^the^decoding^oligonucleotide^in^the^kits^and / or^methods^of^ the^present^disclosure^may^be^a^“multi-decoder”.^A^“multi-decoder”^is^a^decoding^oligonucleotide^ RES-PA13-PCT that^consists^of^at^least^three^sequence^elements.^One^sequence^element^(the^identifier^connector^ element^(t))^can^specifically^bind^to^identifier^oligonucleotide^(o)^of^an^identifier^element^(Z)^of^ the^corresponding^analyte-specific^probe^set^and^at^least^two^other^sequence^elements^(translator^ elements^(c))^specifically^bind^different^signal^oligonucleotides^(each^of^these^sequence^elements^ specifically^binds^a^signal^oligonucleotide^that^differs^to^all^other^signal^oligonucleotides^recruited^ by^other^elements^of^the^multi-decoder).^The^length^of^the^sequence^elements^is^within^the^range^ 8-60^nt,^ preferably^ 12-40^nt,^more^preferably^14-20^nt,^ depending^on^ the^number^of^ analytes^ detected^in^parallel,^the^stability^needed^and^the^number^of^different^signal^oligonucleotides^used.^ The^length^of^the^sequence^elements^may^or^may^not^be^the^same. Therefore,^in^some^advantageous^embodiments,^the^decoding^oligonucleotide^is^a^multi-decoder^ comprising^ - an^identifier^connector^element^(t)^comprising^a^nucleotide^sequence^which^is^essentially^ complementary^to^at^least^a^section^of^the^identifier^oligonucleotide^(o)^of^an^identifier^ element^(Z)^of^the^corresponding^analyte-specific^probe^set,^and - at^ least^ two^ translator^ elements^ (c)^ comprising^ each^ a^nucleotide^ sequence^ allowing^ a^ specific^hybridization^of^a^different^signal^oligonucleotide. Therefore,^ the^ first^ ^ translator^ element^ binds^ a^ different^ signal^ oligonucleotide^ as^ the^ second^ translator^ element.^ In^ particular,^ the^ signal^ oligonucleotides^ differ^ in^ the^ signal^ element^ comprised^in^the^signal^oligonucleotide,^e.g.^in^the^kind^of^the^fluorophore. A^"signal^oligonucleotide"^or^a^“reporter”^as^used^herein^comprises^at^least^two^elements,^a^so- called^“translator^connector^element” (C)^or^"second^connector^element"^(C)^having^a^nucleotide^ sequence^specifically^hybridizable to^at^least^a^section^of^the^nucleotide^sequence^of^the^identifier^ oligonucleotide^(o)^comprised^in^an^identifier^element^(Z)^and / or^the^translator^element^(c)^of^a decoding^oligonucleotide,^and^a^"signal^element"^which^provides^a^detectable^signal and / or an^ additional^ sequence^ that^ can^ be^ detected^ by^ hybridizing^ to^ one^ or^ multiple^ complementary^ oligonucleotides^that^are^labeled.^This^element^can^either^actively^generate^a^detectable^signal^or^ provide^such^a^signal^via^manipulation,^e.g.^fluorescent^excitation.^Typical^signal^elements^are,^for^ example,^enzymes^that^catalyze^a^detectable^reaction,^fluorophores,^radioactive^elements^or^dyes.^ A^ "set"^ refers^ to^ a^ plurality^ of^ moieties^ or^ subjects,^ e.g.^ analyte-specific^ probes,^ signal^ oligonucleotides or^decoding^oligonucleotides,^whether^the^individual^members^of^said^plurality^ RES-PA13-PCT are^identical or^different^from^each^other.^In^all analyte^specific^probe^sets,^the^identical^identifier^ elements (T)^of^the^binding^elements^(Y)^of^the^analyte^specific^probes^are^identical. Therefore, in^ an^ advantageous^ embodiment each^ set^ of^ analyte-specific^ probes comprises^ premixed^complexes,^wherein^the^complexes^of^one^analyte-specific^probe set^compared^to^the^ complexes^of^another^analyte-specific^probe set^comprises^a^different^binding^element^(Y)^that^ specifically^ interacts^with^one^of^ the^different^ analytes^ to^be^ encoded but^ comprises^ identical^ identifier^elements (T) that^interact^with^ identifier^elements (Z) comprising^different identifier^ fluorophores (f)^or^identifier^oligonucleotides (o) compared^to^the^the^identifier^elements (Z) of^ another^set^of^analyte-specific^probes. “Selective^denaturation”^may^be^the^process^of^eliminating^bound^decoding^oligonucleotides^and^ signal^oligonucleotides^with^highest^efficiency^while^at^the^same^time^the^target^specific^probes^ have^ to^stay^hybridized^with^ the^highest^efficiency.^The^ total^efficiency^of^ these^ two^combined^ events^may^to^be^at^least^0.22^for^two^detection^cycles,^0.37^for^three^detection^cycles,^0.47^for^ four^detection^cycles,^0.55^for^five^detection^cycles,^0.61^for^six^detection^cycles,^0.65^for^seven^ detection^cycles,^0.69^ for^eight^detection^cycles,^ 0.72^ for^nine^detection^cycles^and^0.74^ for^10^ detection^cycles,^0.76^for^11^detection^cycles^and^0.78^for^12^detection^cycles. In^an^embodiment^of^the^disclosure a^single^set^refers^to^a^plurality^of^oligonucleotides. An^"analyte^specific^probe^set"^refers^to^a^plurality^of^moieties^or^subjects,^e.g.^analyte-specific^ probes^that^are^different^from^each^other^and^bind^to^independent regions^of^the^analyte.^A^single^ analyte^specific^probe^set^is^further^characterized^by^the^same^identifier^element^(Z). A^"decoding^oligonucleotide^set"^refers^to^a^plurality^of^decoding^oligonucleotides^specific^for^a^ certain^unique^ identifier^needed^to^realize^ the^encoding^independent^of^the^ length^of^ the^code^ word.^Each^and^all^of^the^decoding^oligonucleotides^included^in^a^"decoding^oligonucleotide^set"^ bind^to^the^same^identifier^oligonucleotide^(o)^of^the^analyte-specific^probe. In^certain^embodiments,^this^pattern^of^binding^or^hybridization^of^the^decoding^oligonucleotides^ may^be^converted^into^a^"code^word."^For^example,^the^code^words could^be^also^"101"^and^"110"^ for^an^analyte,^where^a^value^of^1^represents^binding^and^a^value^of^0^represents^no^binding.^The^ RES-PA13-PCT code^words may^also^have^longer^lengths^in^other^embodiments^(see^Fig.^13).^A^code^word can^be^ directly^related^to^a^specific^unique^identifier^sequence^of^a^analyte-specific^probe.^Accordingly,^ different^analyte-specific^probemay^match^certain^code^words,^which^can^then^be^used^to^identify^ the^different^analytes of^the^analyte-specific^probe^based^on^the^binding^patterns^of^the^decoding^ oligonucleotide.^However,^ if^no^binding^ is^evident,^ then^ the^code^word would^be^"000"^ in^ this^ example. The^values^in^each^code^word can^also^be^assigned^in^different^fashions^in^some^embodiments.^For^ example,^a^value^of^0^could^represent^binding^while^a^value^of^1^represents^no^binding.^Similarly,^ a^value^of^1^could^represent^binding^of^a^secondary^nucleic^acid^probe^with^one^type^of^signaling^ entity^while^a^value^of^0^could^represent^binding^of^a^secondary^nucleic^acid^probe^with^another^ type^ of^ distinguishable^ signaling^ entity.^ These^ signaling^ entities^ could^ be^ distinguished,^ for^ example,^via^different^colors^of^ fluorescence.^ In^some cases,^values^ in^code^words need^not^be^ confined^to^0^and^1.^The^values^could^also^be^drawn^from^larger^alphabets,^such^as^ternary^(e.g.,^0,^ 1,^and^2)^or^quaternary^(e.g.,^0,^1,^2,^and^3)^systems.^Each^different^value^could,^for^example,^be^ represented^by a^different^distinguishable^signaling^entity,^ including^(in^some^cases)^one^value^ that^may^be^represented^by^the^absence^of^signal. The^code^words for^each^analytemay^be^assigned^sequentially,^or^may^be^assigned^at^random.^For^ instance,^ ^ a^ first^ analyte may^ be^ assigned^ to^ 101,^ while^ a^ second^ nucleic^ acid^ target^ may^ be^ assigned^to^110.^ In^addition,^ in^some^embodiments,^ the^code^words may^be^assigned^using^an^ error-detection^system^or^an^error- correcting^system,^such^as^a^Hamming^system,^a^Golay^code,^ or^an^extended^Hamming^system^(or^a^SECDED^system,^i.e.,^single^error^correction,^double^error^ detection).^Generally^speaking,^such^systems^can^be^used^to^identify^where^errors^have^occurred,^ and^in^some^cases,^such^systems^can^also^be^used^to^correct^the^errors^and^determine^what^the^ correct^code^word should^have^been.^For^example,^a^code^word such^as^001^may^be^detected^as^ invalid^ and^ corrected^ using^ such^ a^ system^ to^ 101,^ e.g.,^ if^ 001^ is^ not^ previously^ assigned^ to^ a^ different^target^sequence.^A^variety^of^different^error-correcting^codes^can^be^used,^many^of^which^ have^ previously^ been^ developed^ for^ use^ within^ the^ computer^ industry;^ however,^ such^ error- correcting^systems^have^not^typically^been^used^within^biological^systems.^Additional^examples^ of^such^error-correcting^codes^are^discussed^in^more^detail^below. RES-PA13-PCT "Essentially^ complementary"^ means,^ when^ referring^ to^ two^ nucleotide^ sequences,^ that^ both^ sequences^can^specifically^hybridize^to^each^other^under^stringent^conditions,^thereby^forming^a^ hybrid^nucleic^acid^molecule^with^a^sense^and^an^antisense^strand^connected^to^each^other^via^ hydrogen^bonds^(Watson-and-Crick^base^pairs).^"Essentially^complementary"^includes^not^only^ perfect^ base-pairing^ along^ the^ entire^ strands,^ i.e.^ perfect^ complementary^ sequences^ but^ also^ imperfect^complementary^sequences^which,^however,^still^have^the^capability^to^hybridize^to^each^ other^ under^ stringent^ conditions.^ Among^ experts^ it^ is^ well^ accepted^ that^ an^ "essentially^ complementary"^ sequence^ has^ at^ least^ 88%^ sequence^ identity^ to^ a fully^ or^ perfectly^ complementary^sequence. "Percent^sequence^identity"^or^"percent^identity"^in^turn^means^that^a^sequence^is^compared^to^a^ claimed^or^described^sequence^after^alignment^of^the^sequence^to^be^compared^(the^"Compared^ Sequence")^with^ the^ described^ or^ claimed^ sequence^ (the^ "Reference^ Sequence").^ The^ percent^ identity^is^then^determined^according^to^the^following^formula:^percent^identity^=^100^[1^-(C / R)],^ wherein^ C^ is^ the^ number^ of^ differences^ between^ the^ Reference^ Sequence^ and^ the^ Compared^ Sequence^ over^ the^ length^ of^ alignment^ between^ the^ Reference^ Sequence^ and^ the^ Compared^ Sequence,^wherein^ (i)^each^base^or^amino^acid^in^the^Reference^Sequence^that^does^not^have^a^corresponding^aligned^ base^or^amino^acid^in^the^Compared^Sequence^and (ii)^each^gap^in^the^Reference^Sequence^and (iii)^each^aligned^base^or^amino^acid^in^the^Reference^Sequence^that^is^different^from^an^aligned^ base^or^amino^acid^in^the^Compared^Sequence,^constitutes^a^difference^and^(iiii)^the^alignment^has^ to^start^at^position^1^of^the^aligned^sequences; and^R^is^the^number^of^bases^or^amino^acids^in^the^Reference^Sequence^over^the^ length^of^the^ alignment^with^ the^Compared^Sequence^with^ any^ gap^ created^ in^ the^Reference^Sequence^ also^ being^counted^as^a^base^or^amino^acid. If^an^alignment^exists^between^the^Compared^Sequence^and^the^Reference^Sequence^for^which^the^ percent^identity^as^calculated^above^is^about^equal^to^or^greater^than^a^specified^minimum^Percent^ Identity^ then^ the^ Compared^ Sequence^ has^ the^ specified^ minimum^ percent^ identity^ to^ the^ Reference^ Sequence^ even^ though^ alignments^may^ exist^ in^ which^ the^ herein^ above^ calculated^ percent^identity^is^less^than^the^specified^percent^identity. RES-PA13-PCT In^the^"incubation"^steps^as^understood^herein^the^respective^moieties^or^subjects^such^as^probes^ or^oligonucleotide,^are^brought^into^contact^with^each^other^under^conditions^well^known^to^the^ skilled^ person^ allowing^ a^ specific^ binding^ or^ hybridization^ reaction,^ e.g.^ pH,^ temperature,^ salt^ conditions^etc.^Such^steps^may^therefore,^be^preferably^carried^out^in^a^liquid^environment^such^ as^a^buffer^system^which^is^well^known^in^the^art.^ The^"removing"^steps^according^to^the^disclosure may^include^the^washing^away^of^the^moieties^ or^subjects^to^be^removed^such^as^the^probes^or^oligonucleotides^by^certain^conditions,^e.g.^pH,^ temperature,^salt^conditions^etc.,^as^known^in^the^art. It^ is^ understood^ that^ in^ an^ embodiment^ of^ the^method^ according^ to^ the^ present^ disclosure a^ plurality^ of^ analytes^ can^be^ encoded^ simultaneously.^This^ requires^ the^use^of^different^ sets^ of^ analyte-specific^probes^in^step^(A).^The^analyte-specific^probes^of^a^particular^set^differ^from^the^ analyte-specific^probes^of^another^set.^This^means^that^the^analyte-specific^probes^of^set^1^bind^to^ analyte^1,^the^analyte-specific^probes^of^set^2^bind^to^analyte^2,^the^analyte-specific^probes^of^set^ 3^ bind^ to^ analyte^ 3,^ etc.^ However,^ all^ analyte-specific^ probes comprises^ the^ same^ identical^ identifier^element^(T). A^"kit"^is^a^combination^of^individual^elements^useful^for^carrying^out^the^use^and / or^method^of^ the^disclosure,^wherein^the^elements^are^optimized^for^use^together^in^the^methods.^The^kits^may^ also^contain^additional^reagents,^chemicals,^buffers,^reaction^vials^etc.^which^may^be^useful^for^ carrying^ out^ the^ method^ according^ to^ the^ disclosure.^ Such^ kits^ unify^ all^ essential^ elements^ required^ to^work^ the^method^ according^ to^ the^ disclosure,^ thus^minimizing^ the^ risk^ of^ errors.^ Therefore,^such^kits^also^allow^semi-skilled^laboratory^staff^to^perform^the^method^according^to^ the^present^disclosure. The^method^is^particularly^qualified^to^encode,^identify,^detect,^count^or^quantify^analytes^or^single^ analytes^molecules^in^a^biological^sample,^ i.e.^such^as^a^sample^which^contains^nucleic^acids^or^ proteins^as^said^analytes.^It^is^understood^that^the^biological^sample^may^be^in^a^form^as^it^is^in^its^ natural^environment^(i.e.^liquid,^semi-liquid,^solid^etc.),^or^processed,^e.g.^as^a^dried^film^on^the^ surface^of^a^device^which^may^be^re-liquefied^before^the^method^is^carried^out.^ RES-PA13-PCT In^another^embodiment^of the^disclosure prior^to^step^(A)^the^biological^tissue^and / or^biological^ cells are^fixed. For^example,^in^some^embodiments,^the^cell^and / or^the^tissue^is^fixed^prior^ to^introducing^the^probes,^e.g.,^to^preserve^the^positions^of^the^analytes^like^proteins^or^ nucleic^acids^within^the^cell.^Techniques^for^fixing^cells^are^known^to^those^of^ordinary^ skill^ in^ the^ art.^ As^ non-limiting^ examples,^ a^ cell^may^ be^ fixed^ using^ chemicals^ such^ as^ formaldehyde,^ paraformaldehyde,^ glutaraldehyde,^ ethanol,^ methanol,^ acetone, acetic^ acid,^or^the^like.^In^one^embodiment,^a^cell^may^be^fixed^using^Hepes-glutamic^acid^buffer- mediated^organic^solvent^(HOPE). This^measure^has^the^advantage^that^the^analytes^to^be^encoded,^e.g.^the^nuclei^acids^or^ proteins,^are^immobilized^and^cannot^escape.^In^doing^so,^the^analytes^then^prepared^for^ a^better^detection^or^encoding^by^the^method^according^to^the^disclosure.^ In^ yet^ a^ further^ embodiment^ within^ the^ set^ of^ analyte-specific^ probes^ the^ individual^ analyte-specific^probes^comprise^binding^elements^(S1,^S2,^S3,^S4,^S5)^which^specifically^ interact^with^different^sub-structures^of^one^of^the^analytes^to^be^encoded. By^this^measure^the^method^becomes^even^more^robust^and^reliable^because^the^signal^ intensity^ obtained^ at^ the^ end^ of^ the^method or^ a^ cycle,^ respectively,^ is^ increased.^ It^ is^ understood,^that^the^individual^probes^of^a^set^while^binding^to^the^same^analyte^differ^in^ their^binding^position^or^binding^site^at^or^on^the^analyte.^The^binding^elements^S1,^S2,^S3,^ S4,^S5^etc.^of^the^first,^second,^third^fourth,^fifth^etc.^analyte-specific^probes^therefore^bind^ to^or^at^a^different^position^which,^however,^may^or^may^not^overlap. In^some^advantageous embodiments^according^to^the^present^disclosure^at^least^20,^in^particular^ at^least^25,^in^particular^at^least^30^different^analytes^are^detected and / or^quantified^in^a^sample^ in^parallel. For^example,^there^may^be^at^least^5,^at^least^10,^at^least^20,^at least^50,^at^least^75,^at^ least^ 100,^ at^ least^ 300,^ at^ least^ 1,000,^ at^ least^ 3,000,^ at^ least^ 10,000,^ or^ at^ least^ 30,000^ distinguishable^ analyte-specific^ probes^ that^ are^ applied^ to^ a^ sample,^ e.g.,^ simultaneously^ or^ sequentially. RES-PA13-PCT In^some^advantageous embodiments^for^the^multiplexing^twenty^(20)^or^more^different^sets^of^ analyte-specific^probes^ for^ encoding^of^ at^ least^20^different^ analytes^or^more^ are^ required,^ in^ particular^more^than^50,^more^than^100^or^more^than^200.^In^the^multiplexing^methods^of^the^ present^ disclosure,^ in^ particular^ at^ least^ 20^ different^ groups^ of^ analytes^ (e.g.^ mRNA^ moleculespolypeptides / proteins)^i.e.^tags^are^targeted. In^some^advantageous embodiments,^at^least^4^rounds^to^collect^information^for^identification^of^ the^analyte^are^carried^out,^wherein^multiple readout^increases^the^accuracy^of^identification^and^ avoids^ false^ positives.^ The^ unique^ tag^ can^ be^ identified^ by^ various^ techniques,^ including^ hybridization,^ e.g.^ with^ labeled^ probes,^ directly^ or^ indirectly^ or^ by^ sequencing^ (by^ synthesis,^ ligation).^In^particular,^the^identity^of^the^tag^can^be^encoded^with^one^single^signal^(binary^code),^ two^ or^ more^ signals,^ wherein^ the^ signal^ can^ be^ a^ fluorescent^ label^ (e.g.^ attached^ to^ an^ oligonucleotide). In^ some^ advantageous embodiments^ according^ to^ the^ present^ disclosure, the^ kit^ does^ not^ comprise^sets^of^analyte-specific^probes^as^defined^under^item^A). Preferably,^if^the^analyte^in^the^kits^or^methods^according^to^the^present^disclosure^is^a^nucleic^ acid,^each^set^of^analyte-specific^probes^comprises^at^ least^ five^(10)^analyte-specific^probes,^ in^ particular^at^least^fifteen^(15)^analyte-specific^probes,^in^particular^at^least^twenty^(20)^analyte- specific^ probes^ which^ specifically^ interact^ with^ different^ sub-structures^ of^ the^ same^ analyte.^ Nucleic^ acid^ analyte^ includes^ specific^ DNA^ molecules,^ e.g.^ genomic^ DNA,^ nuclear^ DNA,^ mitochondrial^DNA,^viral^DNA,^bacterial^DNA,^extra- or^intracellular^DNA^etc.,^and^specific^mRNA^ molecules,^e.g.^hnRNA,^miRNA,^viral^RNA,^bacterial^RNA,^extra- or^intracellular^RNA,^etc. Preferably,^if^the^analyte^in^the^kits^or^methods^according^to^the^present^disclosure^is^a^peptide,^a^ polypeptide^or^a^protein,^each^set^of^analyte-specific^probes^comprises^at^least^two^(2)^analyte- specific^probes,^in^particular^at^least^three^(3)^analyte-specific^probes,^in^particular^at^least^four^ (4)^analyte-specific^probes^which^specifically^interact^with^different^sub-structures^of^the^same^ analyte.^ RES-PA13-PCT In^some^advantageous embodiments^according^to^the^present^disclosure^the^kit^comprises^at^least^ two^ different sets^ of^ signal^ oligonucleotides,^ wherein^ the^ signal^ oligonucleotides^ in^ each^ set^ comprise^a^different^signal^element^and^comprise^a^different^connector^element^(C). In^particular,^ the^kit^may^comprise^at^ least^ two^different^sets^of^decoding^oligonucleotides^per^ analyte, wherein^ the^decoding^oligonucleotides^comprised^ in^ these^different^sets^comprise^ the^ same^ identifier^ connector^ element^ (t)^ comprising^ a^ nucleotide^ sequence^ which^ is^ essentially^ complementary^to^at^least^a^section^of^the^unique identifier^sequence^of^the^identifier^element^(T)^ of^the^corresponding^analyte-specific^probe^set,^and^wherein^the^decoding^oligonucleotides^of^the^ different^sets^per^analyte^differ^in^the^translator^element^(c)^comprising^a^nucleotide^sequence^ allowing^a specific^hybridization^of^a^signal^oligonucleotide.^ In^some^embodiments^the^kit^comprises^at^least^two^different^sets^of^decoding^oligonucleotides^ per^analyte,^wherein^the^decoding^oligonucleotides^comprised^in^these^different^sets^comprise^the^ same^ identifier^ connector^ element^ (t)^ comprising^ a^ nucleotide^ sequence^ which^ is^ essentially^ complementary^to^at^least^a^section^of^the^unique^identifier^sequence^of^the^identifier^element^(T)^ of^the^corresponding^analyte-specific^probe^set,^and^wherein^the^decoding^oligonucleotides^of^the^ different^sets^for^at^least^one^analyte^differ^in the^translator^element^(c)^comprising^a^nucleotide^ sequence^allowing^a^specific^hybridization^of^a^signal^oligonucleotide.^ In^some^advantageous embodiments,^the^number^of^different^sets^of^decoding^oligonucleotides^ per^analyte^comprising^different^translator^elements^(c)^corresponds^to^the^number^of^different^ sets^ of^ signal^ oligonucleotides^ comprising^ different^ connector^ elements^ (C).^ However,^ the^ decoding^ oligonucleotides^ in^ a^ particular^ set^ of^ decoding^ oligonucleotides^may^ interacts^with^ identical^ identifier^ oligonucleotide^ (o)^ of^ an^ identifier^ element^ (Z)^ which^ are^ unique^ to^ a^ particular^analyte.^ In^particular,^all^ sets^of^decoding^oligonucleotides^ for^ the^different^analytes^ may^comprise^the^same^type(s)^of^translator^element(s)^(c). In^ another^ aspect,^ the^ present^ disclosure^ is generally^ directed^ to^ a^methods^ including^ acts^ of^ exposing^a^sample^to^a^plurality^of^analyte-specific^probes;^for^each^of^the^analyte-specific^probes,^ determining^binding^of^the^analyte-specific^probes^within^the^sample;^creating^code^words based^ on^ the^ binding^ of^ the^ analyte-specific^ probes,^ the^ decoding^ oligonucleotides and^ the^ signal^ oligonucleotides;^and^for^at^least^some^of^the^code^words,^matching^the^code^word to^a^valid^code^ RES-PA13-PCT word.^ In^ certain^ embodiments,^ this^pattern^of^ binding^or^hybridization^of the^ analyte-specific^ probes,^ the^decoding^oligonucleotides and^the^signal^oligonucleotides^may^be^converted^ into^a^ "code^word."^For^example,^for^instance,^the^code^words may^be^"101"^and^"110"^for^a^first^analyte and^ a^ second^ analyte,^ respectively,^ where^ a^ value^ of^ 1^ represents^ binding^ and^ a^ value^ of^ 0^ represents^no^binding^of^decoding^oligonucleotides and / or^the^binding^of^signal^oligonucleotides without^and / or^quenched^signal^element. The^analyte^in^the^detection^round / cycle^is^therefore^ not^detectable^during^imaging. To^create^such^a^zero^(0)^in^a^code^word for^an^individual^analyte^the^kit^may^comprise:^ (D) at^ least^ a^set^ of^non-signal^decoding^oligonucleotides^ for^binding^ to^ a^particular^ identifier^ element^(T)^of^analyte-specific^probes,^wherein^the^decoding^oligonucleotides^in^the^same^set^of^ non-signal^decoding^oligonucleotides^interacting^with^the^same^different^identifier^element^(T), wherein^ each^ non-signal^ decoding^ oligonucleotide^ comprises^ an^ identifier^ connector^ element^(t)^comprising^a^nucleotide^sequence^which^is^essentially^complementary^to^at^least^a^ section^of^a^unique^identifier^sequence,^and^does^not^comprise^a^translator^element^(c)^comprising^ a^nucleotide^sequence^allowing^a^specific^hybridization^of^a^signal^oligonucleotide.^ In^ some^advantageous embodiments,^ all^ steps^ are^ automated,^ in^particular^by^using^ a^ robotic^ system^ and / or^ an^ optical^ multiplexing^ system^ according^ to^ the^ present^ disclosure.^ In^ some^ examples,^the^steps^may^be performed^in^a^fluidic^system. As^mentioned^above,^with^the^methods^according^to^the^present^disclosure^an^encoding^scheme^ with^a^code^word^per^analyte^ is^generated.^Therefore,^ ^each^ analyte^may^be^associated^with^ a^ specific^code^word,^wherein^said^code^word^comprise^a^number^of^positions,^and^wherein^each^ position^corresponds^ to^one^cycle^resulting^ in^a^plurality^of distinguishable^ encoding^schemes^ with^the^plurality^of^code^words. In^particular, said^encoding^scheme^may^be predetermined^and^ allocated^to^the^analyte^to^be^encoded. As^mentioned^above,^the^analyte^to^be^encoded^may^be a^nucleic^acid,^preferably^DNA,^PNA,^RNA,^ in^particular^mRNA,^a^peptide,^polypeptide,^ a^protein or^combinations^ thereof. Therefore,^ the^ binding^ element^ (Y)^may^ comprise^ an^ amino^acid^ sequence^ allowing^ a^ specific^ binding^ to^ the^ analyte^to^be^encoded. Examples for^a^binding^element^(Y)^aremoieties^which^are^affinity^moieties^ from^affinity^substances^or^affinity^substances^in^their^entirety^selected^from^the^group^consisting^ of^antibodies,^antibody^fragments,^anticalin^proteins,^receptor^ligands,^enzyme^substrates,^lectins,^ RES-PA13-PCT cytokines,^ lymphokines,^ interleukins,^ angiogenic^ or^ virulence^ factors,^ allergens,^ peptidic^ allergens,^ recombinant^ allergens,^ allergen-idiotypical^ antibodies,^ autoimmune-provoking^ structures,^ tissue-rejection-inducing^ structures,^ immunoglobulin^ constant^ regions^ and^ combinations^thereof. In^particular,^the^binding^element^(Y is^an^antibody^or^an^antibody^fragment^ selected^from^the^group^consisting^of^Fab,^scFv;^single^domain,^or^a^fragment^thereof,^bis^scFv,^Fab^ 2,^Fab^3,^minibody,^diabody,^triabody,^tetrabody^and^tandab. By^this^measure^the^method^is^further^developed^to^such^an^extent^that^the^encoded^analytes^can^ be^detected^by^any^means^which^is^adapted^to^visualize^the^signal^element.^Examples^of^detectable^ physical^features^include^e.g.^light,^chemical^reactions,^molecular^mass,^radioactivity,^etc. In^ some^ advantageous embodiments,^ the^ signal^ caused^ by^ the^ signal^ element,^ therefore^ in^ particular^the^binding^of^the^signal^oligonucleotides^to^the^decoding^oligonucleotides,^interacting^ with^the^corresponding^analyte^probes,^bound^to^the^respective^analyte^is^determined^by: (a) Imaging^at^least^a^portion^of^the^sample;^and / or (b) Using^an^optical^imaging^technique;^and / or (c) Using^a^fluorescence^imaging^technique;^and / or (d) Multi-color^fluorescence^imaging^technique;^and / or^ (e) Super-resolution^fluorescence^imaging^technique. The^kits^and^method^according to^the^present^disclosure^may^be^used^ideally^for^in^vitromethods^ for^ diagnosis^ of^ a^ disease^ selected^ from^ the^ group^ comprising^ cancer,^ neuronal^ diseases,^ cardiovascular^diseases,^inflammatory^diseases,^autoimmune^diseases,^diseases^due^to^a^viral^or^ bacterial^infection,^skin^diseases,^skeletal^muscle^diseases,^dental^diseases^and^prenatal^diseases. Further,^the^kits^and^method^according to^the^present^disclosure^may^be^used^also^ideally^ for^ in^ vitro methods^ for^ diagnosis^ of^ a^ disease^ in^ plants^ selected^ from^ the^ group^ comprising:^ diseases^ caused^ by^ biotic^ stress,^ preferably^ by^ infectious^ and / or^ parasitic^ origin,^ or^ diseases^ caused^ by^ abiotic^ stress,^ preferably^ caused^ by^ nutritional^ deficiencies^ and / or^unfavorable^environment. RES-PA13-PCT Further,^the^kits^and^method^according to^the^present^disclosure^may^be^used^also^ideally^for^in^ vitro methods^ for^ screening,^ identifying^ and / or^ testing^ a^ substance^ and / or^ drug^ comprising: (a) contacting^a^test^sample^comprising^a^sample^with^a^substance^and / or^drug^ (b) detecting^different^analytes^in^a^sample^by^sequential^signal-encoding^of^said^ analytes^with^a^method^according^to^the^present^disclosure. An^ optical^ multiplexing^ system^ suitable^ for^ the^ method^ according^ to^ the^ present^ disclosure,^ comprising^at^least: - a reaction^vessel^for^containing^the^kits^or^part^of^the^kits^according^to^the^present^ disclosure; - a^detection^unit^comprising^a^microscope,^in^particular^a^fluorescence^microscope; - a^camera; - a^liquid^handling^device. In^ some^ embodiments, optical^multiplexing^ system^may^ comprises^ further^ a^ heat^ and^ cooling^device and / or^a^robotic^system. In^some^advantageous embodiments,^the^method^described^herein^is^used^for^specific^detection^ of^many^different^analytes^in^parallel.^The^technology^allows^to^distinguish^a^higher^number^of^ analytes^than^different^signals^are^available.^The^process^includes^at^least^four^consecutive^rounds^ of^ specific^ binding,^ signal^ detection^ and^ selective^ denaturation^ (if^ a^ next^ round^ is^ required),^ eventually^producing^ a^ signal^ code.^To^decouple^ the^dependency^between^ the^ analyte^ specific^ binding^ and^ the^ oligonucleotides^ providing^ the^ detectable^ signal,^ a^ so^ called^ “decoding”- oligonucleotide^ is^ introduced.^The^decoding^oligonucleotide^ transcribes^ the^ information^of^ the^ analyte^specific^probe^set^to^the^signal^oligonucleotides.^ RES-PA13-PCT The^disclosure is^now^further^explained^by^means^of^embodiments^resulting^in^additional^features, characteristics^and^advantages^of^the^disclosure.^The^embodiments^are^of^pure^illustrative^nature^ and^ do^ not^ limit^ the^ scope^ or^ range^ of^ the^ disclosure.^ The^ features^mentioned^ in^ the^ specific^ embodiments^are^general^features^of^the^disclosure which^are^not^only^applicable^in^the^specific^ embodiment^but^also^in^an^isolated^manner^in^the^context^of^any^embodiment^of^the^disclosure. The^method^disclosed^herein^is^used^for^specific^detection^of^many^different^analytes^in^parallel.^ The^ technology^ allows^ distinguishing^ a^ higher^ number^ of^ analytes^ than^ different^ signals^ are^ available.^The^process^preferably^ includes^ at^ least^ two^ consecutive^ rounds^of^ specific^ binding,^ signal^detection^and^selective^denaturation^(if^a^next^round^is^required),^eventually^producing^a^ signal^ code.^ To^ decouple^ the^ dependency^ between^ the^ analyte^ specific^ binding^ and^ the^ oligonucleotides^ providing^ the^ detectable^ signal,^ a^ so^ called^ “decoding”^ oligonucleotide^ is^ introduced.^ The^ decoding^ oligonucleotide^ transcribes^ the^ information^ of^ the^ analyte^ specific^ probe^set^to^the^signal^oligonucleotides. The^present^disclosure^pertains^further^to^methods^of^detecting^an^analyte,^comprising:^ - attaching^ a^ plurality^ of^ premixed^ analyte-specific^ probes^ to^ the^ analyte,^ wherein^ the^ analyte-specific^ probes^ independently^ attach^ to^ the^ analyte^ and^ wherein^ the^ analyte- specific^ probes in^ all^ sets share^ a^ identical^ identifier^ element (T) and^ comprises^ an^ identifier^element^(Z)^that^specifically^interact^and^bind^to^the^identifier^element^(T)^of^the^ binding^ element^ (Y),^ wherein^ the^ identifier^ element^ (Z)^ is^ coupled^ to^ an^ identifier^ fluorophore^(f)^or^an^identifier^oligonucleotide^(o)^that^is^used^to^identify^the^identity^of^ the^ identifier^ element^ (Z),^ wherein^ the^ identifier^ fluorophore^ (f)^ or^ the^ identifier^ oligonucleotide^ (o)^ of^ the^ identifier^ element^ (Z)^ in^ a^ particular^ set^ of^ analyte-specific^ probes^differ^from^the^identifier^fluorophore^(f)^or^the^identifier^oligonucleotide^(o)^of^the^ identifier^element^(Z)^of^another^set^of^analyte-specific^probes; - detecting^ the^ signal^ caused^ by^ the^ identifier^ fluorophore^ (f)^ coupled^ to^ the^ identifier^ element^(Z);^or RES-PA13-PCT - annealing^signal^oligonucleotides to^to^the^identifier^oligonucleotide^(o)^of^the^identifier^ element^ (Z) comprised^ in^ the^ analyte-specific^ probe complexes; hybridizing^ one^ or^ multiple^complementary^oligonucleotides^to^the^signal^oligonucleotides^that^are^labeled - Detecting the^signals caused^by^the^signal^elements^of^the^signal^oligonucleotides or^by^the^ complementary^oligonucleotides,^or - annealing^a^plurality^of^first^decoding^oligonucleotides^to^the^analyte-specific^probes;^ - contacting^the^sample^with^at^least^a^set^of^signal^oligonucleotides - detecting^the^signal^oligonucleotides; - removing^the^plurality^of^first^decoding^oligonucleotides; - annealing^a^plurality^of^second^decoding^oligonucleotides^to^the^analyte-specific^probes; - annealing^ a^ second^ signal^ oligonucleotide^ to^ at^ least^ one^ of^ the^ plurality^ of^ second^ decoding^oligonucleotides.^ In^ summary,^ in^ particular^ the^ present^ disclosure^ pertains^ to the^ combinatorial^ use^ of^ primary^antibodies^together^with^secondary^antibodies,^FABs^or^proteins^that^specially^ bind^ to^ the^ primary^ antibodies or^ proteins that^ recognize^ and^ bind^ specially^ another^ protein. The^analyte^is^in^particular a^protein,^a^nucleic^acid,^or^another^molecule^that^specifically^ is^ recognized^ and^ bound^ by^ the^ binding^ element^ (Y).^ An^ analyte may^ be^ part^ of^ a^ eucaryotic,^ prokaryotic,^ animal,^ fungi,^ plant^ cell,^ an^ organelle,^ or^ an^ extracellular^ area^ within^ an^ organism.^ It^ can^ be^ part^ of^ an^ intact^ or^ disintegrated^ organisms^ or^ cell.^ Disintegration^ of^ an^ organism:^ is^ defined^ as^ any^ type^ of^ surgery,^ extraction,^ lysis,^ purification^or^other^methods^that^disintegrate^the^organism^or^cell. RES-PA13-PCT The^binding^element^(Y) is^defined^in^general^as^a^protein^or^nucleic^acid^that^specifically^ recognizes^and^binds^another^molecule^X.^Y^can^be^an^antibody,^a^fragment^of^an^antibody,^ e.g.,^FAB^or^F(ab)2,^any^other^proteins,^aptamers^or^nucleic^acids^that^specifically^interact^ and^bind^to^X.^ The^identifier^element^(Z)^ is^defined^in^general^as^a^protein^or^nucleic^acid^that^specifically^ recognizes^and^binds^to^Y.^Z^can^be^an^antibody,^a^fragment^of^an^antibody,^e.g.,^FAB^or^ F(ab)2,^any^other^proteins,^aptamers^or^nucleic^acids^that^specifically^interact^and^bind^to^ Y.^In^addition,^Z^is^coupled^to^an^identifier^fluorophore^(f)^or^identifier^(o)^oligonucleotide^ that^is^used^to^identify^the^identity^of^Z.^The^same^type^of^Z^can^be^coupled^to^different^ oligonucleotides^identifiers^(T)^to^get^a^library^of^Zs^coupled^to^different^oligonucleotides.^ The^ library^may^have^a^size^of^at^ least^5,^10,^20,^40,^80^or^more^than^80^Zs^coupled^to^ different^identifier^oligonucleotides.^ The^process^is^divided^in^several^steps: 1) Preforming^ at^ least^ 4^ different^ complexes^ of^ individual^ Ys^ and^ individual^ Zs,^ wherein^Z^is^coupled^to^a^certain^oligonucleotide^to^form^4 different^combinations^ such^as^ a) Y1-Z(identfier-oligoT1) b) Y2-Z(identfier-oligoT2) c) Y3-Z(identfier-oligoT3) d) Y4-Z(identfier-oligoT4) Time,^ temperature,^ concentration,^ and^ other^ conditions^ may^ vary^ depending^ on^ the^ various^Ys^and^Zs) 2) Bind^ the^ complexes^ to^ the^analyte (time,^ temperature,^ concentration,^ and^other^ conditions^may^vary^depending^on^ the^application).^Thereafter,^washing^ steps^may^ be^ required^to^reduce^the^number^of^unspecific^binding^events. RES-PA13-PCT 3) Detect^ the^ Y-Z^ complexes^ bound^ to^ the^ analyte by^ hybridizing^ to^ the^ oligonucleotide^coupled^to^Z.^Thereafter,^washing^steps^may^be^required^to^reduce^the^ number^ of^ unspecific^ binding^ events.^ Various^ options^ are^ possible^ to^ identify^ the^ identifier: a) Identifier^may^hybridize^to^a^labeled^oligonucleotide^that^is^complementary^to^the^ sequence^of^the^identifier.^The^label^may^be^a^fluorophore,^an^enzyme,^or^another^label^ that^can^be^detected. b) Identifier^may^hybridize^to^a^non-labeled^oligonucleotide^that^is^complementary^to^ the^sequence^of^the^identifier.^This^oligonucleotide^has^an^addition^sequence^that^can^be^detected^ by^hybridizing^to^one^or^multiple^complementary^oligonucleotides^that^are^labeled.^ c) The^identification^of^the^identifier^may^require^several^rounds^to^build^up^a^code^word. The^process^may^require^additional^steps^for^complex^formation^such^as^filtration,^purification,^ absorption,^ size^ selection^ etc.^ to^purify^proper^ complexes^ from^ individual^ components^or^ side^ products.^ In^particular,^the^methods^of^the^present^disclosure^may^be^used^to^identify^regions^of^interest,^in^ particular^in^an^organ,^tissue^or^single^cell. In^some^advantageous^embodiments,^the^methods^of^the^present^disclosure^can^be^used^for^image^ analysis.^ In^ this^ context,^ the^ methods^ of^ the^ present^ disclosure^ are^ used^ to^ develop^ cell^ segmentation^ algorithms^ using^ immunofluorescence^ images.^ The^ methods^ of^ the^ present^ disclosure^could^be^used^to^aid^the^segmentation^of^cells^in^a^sample,^in^particular^in^a^tissue.^Cell^ segmentation^is^a^crucial^step^since^errors^in^identifying^cells^and^their^boundaries^have^a^direct^ impact^ on^ the^ ability^ to^ correctly^quantify^ the^ expression^ levels^ of^ proteins^ in^ these^ cells.^ For^ example,^ information^ about^ the^ levels^ of^ proteins^ in^ single^ cells^ can^ also^ be^ used^ to^ answer^ questions^regarding^the^set^of^cell^types^in^a^sample,^the^spatial^distribution^of^these^types,^and^ the^interactions^between^different^cells^and^cell^types^in^a^tissue. RES-PA13-PCT A^unique^advantage^of^spatial^proteomics^is^that,^unlike^prior^methods,^they^do^not^require^specific^ markers^to^be^selected^in^advance.^Since^these^studies^profile^tens^of^proteins,^a^marker^can^be^ selected^post-experiment.^Moreover,^there^is^no^need^to^select^a^single^marker.^Even^if^a^single^ marker^does^not^work^for^all^cells,^a^combination^of^such markers^may^be^useful^for^segmenting^ all^cells. Furthermore,^ the^ methods^ of^ the^ present^ disclosure^ provide^ spatial^ information^ about^ the^ location^ of^ the^ cells^ being^ profiled^ enabling^ the^ analysis^ of^ cell-cell^ signaling^ and^ cell^ type^ organization. By^using the^methods^according^to^the^present^disclosure^of^detecting^analytes^like^biomolecules^ in^a^ fixed^biological^sample,^ up^ to^ three^analytes^ like^proteins^on^ the^surface^of^ cells^could^be^ detected^thereby^visualizing^the^boundaries^of^cells.^^The^analyte^may^be^bound^by^an^antibody,^ an^antibody^ fragment,^an^aptamer^or^a^nucleic^acid^ that^specifically^ interacts^and^binds^to^ the^ analyte,^wherein^in^particular^the^antibody^fragment^is^selected^from^the^group^consisting^of^a^ Fab,^ scFv,^ single^ domain,^ bis^ scFv,^ Fab2,^ Fab3,^ minibody,^ diabody,^ triplebody,^ tetrabody^ and^ tandab. In^particular,^the^analyte^to^be^detected^may^be^Sodium-potassium^ATPase,^plasma^membrane^ calcium^ATPase^ (PMCA),^ Cadherin,^ CD98,^ Caveolae,^ HER2,^ Beta-Catenin,^ CAH9,^ E-Cadherin,^ c- MYC,^Vimentin,^among^others.^^The^choice^of^the^binding^elements^like^antibodies^will^vary^based^ on^the^cell^type^to^be^detected.^^Each^tissue^has^a^different^composition^of^cell^types.^^By^visualizing^ the^boundaries^of^cells,^a^user^could^by^eye^or^through^a^computational^tool^segment^cell. In^some^further^advantageous^embodiments,^the^methods^of^the^present^disclosure^may^be^used^ to^ establish^ AI-based^ image^ analysis^ algorithm^ using^ immunofluorescence^ images^ to^ identify^ disorders.^Furthermore,^the^advantageous^embodiments^the^methods^of^the^present^disclosure^ may^be^used^for^feature^extraction^like^nucleus^morphology,^signal^homogeneity,^signal^intensity^ and / or^centroids.^For^example,^the^morphology^of^cells^often^reflects^their^tissue-specific^function^ or^state.^Cells^undergo^morphological^changes^to^become^motile^in^response^to^various^stimuli,^ either^ as^part^ of^ normal^physiology^ and^development,^ or^due^ to^pathologic^disorder.^ In^ some^ cancers,^epithelial^cells^lose^their^inter-cellular^connections,^and^take^on^protrusive,^exploratory^ morphologies^as^they^transit^from^static^to^migratory^phenotypes.^Recognizing^such^abnormal^cell^ shapes^can^aide^in^properly^identifying^malignant^(as^distinct^from^benign)^tumors.^ RES-PA13-PCT A^ further^ application^ for^ the^methods^of^ the^present^disclosure^ is^ to^determine^ expression^of^ proteins^ and^ genes^ of^ interest^ via^ semi-quantitative^ approaches.^ Semi-quantitative^ immunohistochemistry^ (IHC)^ for^ example^ is^ a^ powerful^ method^ for^ investigating^ protein^ expression^and^localization^within^tissues. In^some^further^advantageous^embodiments,^the^methods^of^the^present^disclosure^may^be^used^ for^diagnosis^and^prognosis^applications.^In^particular,^the^methods^of^the^present^disclosure^are^ used^for^the^determination^of^the^distribution^of^a^target^protein^in^the^of^health^and^disease^organ,^ tissue^and / or^cell.^Furthermore,^the^methods^of^the^present^disclosure^may^be^used^to^predict^the^ prognosis^of^diseases^by^using^disease-related^prognostic^and / or^predictive^biomarkers.^A^variety^ of^factors^influence^a patient’s^clinical^outcome,^including^intrinsic^characteristics^of^the^patient,^ disease,^or^medical^condition,^and^the^effects^of^any^treatments^that^the^patient^receives.^Some^of^ the^intrinsic^characteristics^may^be^reflected^as^prognostic^biomarkers,^i.e.,^biomarkers^used^to^ identify^likelihood^of^a^clinical^event,^disease^recurrence^or^progression^in^patients^who^have^the^ disease^or^medical^condition^of^interest,^and^others^as^predictive^biomarkers,^i.e.,^biomarkers^used^ to^ identify^ individuals^who^are^more^ likely^ than^ similar^ individuals^without^ the^biomarker^ to^ experience^ a^ favorable^ or^ unfavorable^ effect^ from^ exposure^ to^ a^ medical^ product^ or^ an^ environmental^agent. In^some^further^advantageous^embodiments,^the^methods^of^the^present^disclosure^may^be^used^ to^provide^disease^diagnosis^e.g.^tumor^of^unknow^origin,^neurodegenerative^disorders,^muscle^ disorders,^brain^trauma,^infectious^diseases.^In^particular,^the^methods^of^the^present^disclosure^ are^used^to^predict^a^therapeutic^response.^A^further^application^of^the^methods^of^the^present^ disclosure^ is^ the^ subclassification^ of^ diseases.^ Classifications^ of^ diseases^ become^ extremely^ important^ in^ the^compilation^of^statistics^on^causes^of^ illness^ (morbidity)^and^causes^of^death^ (mortality).^ Furthermore,^the^methods^of^the^present^disclosure^may^be^used^to^examine^gene^expression^and^ to^study^gene^regulation^in^normal^and^pathological^tissues. In^some^further^advantageous^embodiments,^the^methods^of^the^present^disclosure^may^be^used^ for^morphological^studies.^In^particular,^the^methods^of^the^present^disclosure^are^used^for^the^ recognition^of^ tissue^structures^and / or^cell^ type^classifications.^ In^ this^context,^a^cell^ type^ is^ a^ classification^ used^ to^ identify^ cells^ that^ share^ morphological^ or^ phenotypical^ features.^ A^ RES-PA13-PCT multicellular^organism^may^contain^cells^of^a^number^of^widely^differing^and^specialized^cell^types,^ such^as^muscle^cells^and^skin^cells,^that^differ^both^in^appearance^and^function^yet^have^identical^ genomic^sequences.^Cells^may^have^the^same^genotype,^but^belong^to^different^cell^types^due^to^ the^differential^regulation^of^the^genes^they^contain.^Classification^of^a^specific^cell^type^is^often^ done^through^the^use^of^microscopy^(such^as^those^from^the^cluster^of^differentiation^family^that^ are^commonly^used^for^this^purpose^in^immunology). In^some^further^advantageous^embodiments,^the^methods^of^the^present^disclosure^may^be^used^ for^multiomics^studies.^Multiomics^ is^a^biological^analysis^approach^ in^which^ the^data^sets^are^ multiple^ "omes", such^ as^ the^ genome,^ proteome,^ transcriptome,^ epigenome,^metabolome,^ and^ microbiome.^By^combining^these^"omes",^scientists^can^analyze^complex^biological^big^data^to^find^ novel^associations^between^biological^entities,^pinpoint^relevant^biomarkers^and^build^elaborate^ markers^of^disease^and^physiology.^In^doing^so,^multiomics^integrates^diverse^omics^data^to^find^ a^ coherently^ matching^ geno-pheno-envirotype^ relationship^ or^ association.^ In^ particular,^ the^ methods^ of^ the^ present^ disclosure^ are^ used^ for^ the^ quantitative^ correlation^ and^ spatial^ colocalization^study^between^ISH^and^ICH.^As^mentioned^above,^immunohistochemistry^(IHC)^is^ an^invaluable^tool^for^the^detection,^localization,^and^quantification^of^antigens^in^preserved^tissue^ for^research^and^diagnostic^purposes.^In^situ^hybridization^(ISH)^is^a^unique^molecular^analysis^ method^because^it^provides^the^precise^microscopic^localization^of^analytes^like^proteins,^nucleic^ acids^such^as^DNA,^mRNA^and^microRNA^in^metaphase^spreads^and^cell^and^tissue^preparations.^ In^particular,^the^methods^of^the^present^disclosure^are^used^for^cell^type^classification^based^on^ such^multi-omics^data.^In^a^further^embodiment,^the^methods^of^the^present^disclosure^are^used^ to^ study^ epigenetic^modifications^ and^ chromatin^ structures.^ In^ particular,^ the^methods^ of^ the^ present^ disclosure^ are^ used^ to^ identify^ spatial^ distribution,^ and^ cell-specific^ ISH^ signals^ (Cell^ segmentation).^ In^some^further^advantageous^embodiments,^the^methods^of^the^present^disclosure^may^be^used^ to^determine the^correlation^of^genetic^aberrations^and / or^the^copy^number^variations^(CNV)^to^ the^corresponding^proteins,^wherein^CNV^is^a^phenomenon^in^which^sections^of^the^genome^are^ repeated^and^the^number^of^repeats^in^the^genome^varies^between^individuals. Therefore,^the^present^disclosure^pertains^to^the^following^items: RES-PA13-PCT 1. A^method^for^detecting^different^analytes^in^a^sample^simultaneously^comprising^the^steps^ of:^ (A) contacting^the^sample^with^at^least^four^(4)^different^sets^of^analyte-specific^probes^for^ detecting^of^at^ least^4^different^analytes,^each^set^of^analyte-specific^probes^ interacting^ with^a^different^analyte,^wherein^each^analyte-specific^probe^is^a^complex^comprising (aa)^a^binding^element^(Y)^that^specifically^interacts^with^one^of^the^different^ analytes^ to^ be^ encoded,^wherein^ each^ binding^ element^ (Y)^ of^ each^ set^ of^ analyte-specific^probes^comprises^an^identical^identifier^element^(T),^and (bb)^ an^ identifier^ element^ (Z)^ that^ specifically^ interact^ and^ bind^ to^ the^ identifier^ element^ (T)^ of^ the^ binding^ element^ (Y),^ wherein^ the^ identifier^ element^ (Z)^ is^ coupled^ to^ an^ identifier^ fluorophore^ (f)^ or an^ identifier^ oligonucleotide^ (o)^ that^ is^ used^ to^ identify^ the^ identity^ of^ the^ identifier^ element^ (Z),^ wherein^ the^ identifier^ fluorophore^ (f)^ or^ the^ identifier^ oligonucleotide^(o)^of^the^identifier^element^(Z)^in^a^particular^set^of^analyte- specific^ probes^ differ^ from^ the^ identifier^ fluorophore^ (f)^ or^ the^ identifier^ oligonucleotide^(o)^of^ the^ identifier^element^(Z)^of^another^set^of^analyte- specific^probes, wherein^the^binding^element^(Y)^and^the^identifier^element^(Z)^are^premixed^for^ each^set^of^analyte-specific^probes^in^a^separate^reaction^tube^to^form^complexes^between^ the^binding^element^(Y)^and^the^identifier^element^(Z)^before^contacting^the^sample^with^ the^different^sets^of^analyte-specific^probes,^wherein^the^identifier^element^(Z)^interacts^ and^binds^to^the^identical^identifier^element^(T)^of^the^binding^element^(Y), (B) detecting^the^signal^caused^by^the^identifier^fluorophore^(f)^coupled^to^the^identifier^ element^(Z);^or (C)^ i)^ contacting^ the^ sample^ with^ different^ set^ of^ signal^ oligonucleotides,^ each^ signal^ oligonucleotide^comprising: RES-PA13-PCT (aa)^a^translator^connector^element^(C)^comprising^a^nucleotide^sequence^ which^ is^essentially^complementary^ to^at^ least^a^section^of^ the^nucleotide^ sequence^ of^ an^ identifier^ oligonucleotide^ (o)^ comprised^ in^ an^ identifier^ element^(Z),^and (bb)^a^signal^element^and / or^an^additional^sequence^that^can^be^detected^by^ hybridizing^ to^ one^ or^ multiple^ complementary^ oligonucleotides^ that^ are^ labeled^or ii)^contacting^the^sample^with^at^least^one^set^of^decoding^oligonucleotides^per^analyte,^ wherein^in^each^set^of^decoding^oligonucleotides^for^an^individual^analyte^each^decoding^ oligonucleotide^comprises: (aa)^an^identifier^connector^element^(t)^comprising^a^nucleotide^sequence^ which^ is^ essentially^ complementary^ to^ at^ least^ a^ section^ of^ the^ identifier^ oligonucleotide^(o)^of^an^identifier^element^(Z)^of^the^corresponding^analyte- specific^probe^set,^and (bb)^a^translator^element^(c)^comprising^a^nucleotide^sequence^allowing^a^ specific^hybridization^of^a^signal^oligonucleotide; wherein^the^decoding^oligonucleotides^of^a^set^for^an^individual^analyte^differ^from^ the^ decoding^ oligonucleotides^ of^ another^ set^ for^ a^ different^ analyte^ in^ the^ first^ connect^ element^ (t);^ and^ contacting^ the^ sample^ with^ at^ least^ a^ set^ of^ signal^ oligonucleotides,^each^signal^oligonucleotide^comprising: (aa)^a^translator^connector^element^(C)^comprising^a^nucleotide^sequence^ which^ is^essentially^complementary^ to^at^ least^a^section^of^ the^nucleotide^ sequence^ of^ a^ translator^ element^ (c)^ comprised^ in^ a^ decoding^ oligonucleotide,^and (bb)^a^signal^element^and / or^an^additional^sequence^that^can^be^detected^by^ hybridizing^ to^ one^ or^ multiple^ complementary^ oligonucleotides^ that^ are^ labeled; (D)Detecting^the^signal^caused^by^the^signal^element^of^section^C)^i)^and / or^C)^ii);^and^ optionally (E) Selectively^ removing^of^ the^ signal^ oligonucleotides^of^ section^C)^ i)^ or^ the^decoding^ oligonucleotides^ and^ signal^ oligonucleotides^ of^ of^ section^ C)^ ii)^ from^ the^ sample,^ RES-PA13-PCT thereby^essentially^maintaining^the^specific^binding^of^the^analyte-specific^probes^to^ the^analytes^to^be^encoded;^and^optionally (F) Performing^at^least^three^(3)^further^cycles^comprising^steps^C)^to^E)^to^generate^an^ encoding^scheme^with^a^code^word^per^analyte,^wherein^in^particular^the^last^cycle^ may^stop^with^step^(D). 2. The^method^according^to^ item 1,^wherein^the^sample^ is^a^biological^sample,^preferably^ comprising^ biological^ tissue^ from^ human,^ animals,^ or^ plants,^ further^ preferably^ comprising^biological^cells^and / or^extracts^and / or^part^of^cells. 3. The^method^to^any^one^of^items^1^to^2,^wherein^the^cell^is^a^prokaryotic^cell^or^a^eukaryotic^ cell,^an^animal^cell,^a^plant^cell,^ in^particular^a^cell^ from^vertebrate^cells,^ in^particular^a^ mammalian^cell,^in^particular^a^human^cell. 4. The^method^according^to^item 2,^wherein^the^biological^tissues,^biological^cells,^extracts^ and / or^part^of^cells^are^fixed.^ 5. The^method^according^ to^ any^one^of^ items^ 1^ to^4,^wherein^ the^ analytes^ are^ fixed^ in^ a^ permeabilized^sample,^such^as^a^cell-containing^sample. 6. The^method^according^to^any^one^of^items^1^to^5,^wherein^the^analyte^is^a^nucleic^acid,^ preferably^DNA,^PNA^or^RNA,^in^particular^mRNA. 7. The^ method^ according^ to^ any^ one^ of items^ 1^ to^ 5,^ wherein^ the^ analyte^ is^ a^ peptide,^ polypeptide^or^a^protein. 8. The^method^according^to^any^one^of^items^1^to^7,^wherein^the^binding^element^(Y)^is^a^ protein^and / or^a^nucleic^acid^that^specifically^recognizes^and^binds^to^the^analyte. 9. The^method^according^to^any^one^of^items^1^to^8,^wherein^the^binding^element^(Y)^is^an^ antibody,^an^antibody^fragment,^an^aptamer^or^a^nucleic^acid^that^specifically^interacts^and^ RES-PA13-PCT binds^to^the^analyte, wherein^in^particular^the^antibody^fragment^is^selected^from^the^group^ consisting^of^a^Fab,^scFv,^single^domain,^bis^scFv,^Fab2,^Fab3,^minibody,^diabody,^triplebody,^ tetrabody^and^tandab. 10. The^method^according^to^any^one^of^items^1^to^9,^wherein^the^identifier^element^(Z)^is^an^ antibody,^an^antibody^fragment, an^aptamer^or^a^nucleic^acid^that^specifically^interacts^and^ binds^to^the^identifier^element^(Z), wherein^in^particular^the^antibody^fragment^is^selected^ from^ the^ group^ consisting^ of^ a^ Fab,^ scFv,^ single^ domain,^ bis^ scFv,^ Fab2,^ Fab3,^ minibody,^ diabody,^triplebody,^tetrabody^and^tandab. 11. The^method^according^to^any^one^of^items^1^to^10,^wherein^the^identifier^element^(Z)^is^an^ antibody^fragment selected^from^the^group^consisting^of^a^Fab,^scFv,^single^domain,^bis^scFv,^ Fab2,^Fab3,^minibody,^diabody,^triplebody,^tetrabody^and^tandab,^wherein^^in^particular^the^ antibody^fragment^is^a^Fab. 12. The^method^ according^ to^ any^ one^ of^ items^ 1^ to^ 11,^ wherein^ the^ analyte^ is^ a^ protein^ comprised^in^a^biological^sample,^the^binding^element^(Y)^is^an^antibody^or^an^antibody^ fragment^comprising^a^(Fc)^region^and^the^identifier^element^(Z)^is^a^secondary^antibody,^ a^Fab,^Fab2and / or^a^Fab3^fragment.^ 13. The^method^according^to^any^one^of^items^1^to^12,^wherein^after^step^A)^and^before^step^ B)^ the^ identifier^ elements^ (Z)^ are^ blocked^ to^ prevent^ cross^ reactivity,^ in^ particular^ by^ mixing^with^the^binding^element^specific^for^the^identifier^element^(Z).^ 14. The^method^according^to^any^one^of^items^1^to^13,^wherein^after^step^A)^and^before^step^ B)^the^non-bound^analyte-specific^probes^are^removed,^in^particular^by^washing. 15. The^method^according^to^any^one^of^items^1^to^14,^wherein^all^steps^are^automated,^in^ particular^by^using^a^robotic^system. 16. The^method^according^to^any^one^of^items^1^to^15,^wherein^all^steps^are^performed^in^a^ fluidic^system. RES-PA13-PCT 17. The^method^according^to^any^one^of^items^1^to^16,^wherein^each^analyte^is^associated^with^ a^ specific^ code^ word,^ wherein^ said^ code^ word^ comprise^ a^ number^ of^ positions,^ and^ wherein^each^position^corresponds^to^one^cycle^resulting^in^a^plurality^of^distinguishable^ encoding^schemes^with^the^plurality^of^code^words. 18. The^method^ according^ to^ any^ one^ of^ items^ 1^ to^ 17,^wherein^ said^ encoding^ scheme^ is^ predetermined^and^allocated^to^the^analyte^to^be^encoded. 19. The^method^according^to^any^one^of^items^1^to^18,^wherein^the^code^words^obtained^for^ the^ individual^ analytes^ in^ the^ performed^ cycles^ comprise^ the^ detected^ signals^ and^ additionally^at^least^one^element^corresponding^to^no^detected^signal. 20. The^method^according^to^any^one^of^claims^1^to^19,^wherein^no^signal^is^detected^for^at^ least^one^analyte^within^at^least^one^cycle. 21. The^method^according^to^any^one^of^items 1^to^20,^wherein^for^at^least^for^one^individual^ analyte^a^position^of^the^code^word^is^zero^(0). 22. The^method^according^to^any^one^of^items^1^to^21,^wherein^the^code^word^zero^(0[CK1])^is^ generated^by^using^no^decoding^oligonucleotides^having^an^identifier^connector^element^ (t)^ comprising^ a^nucleotide^ sequence^which^ is^ essentially^ complementary^ to^ at^ least^ a^ section^of^the^unique^identifier^sequence^of^the^identifier^element^(T)^of^a^corresponding^ analyte-specific^probe^for^an^individual^analyte. 23. The^method^according^to^any^one^of^items^1^to^21,^wherein^if^at^least^for^one^individual^ analyte^a^position^of^the^code^word^is^zero^(0)^in^this^cycle^no^corresponding^decoding^ oligonucleotides^ having^ an^ identifier^ connector^ element^ (t)^ comprising^ a^ nucleotide^ sequence^which^is^essentially^complementary^to^at^least^a^section^of^the^unique^identifier^ sequence^of^ the^ identifier^element^(T)^of^a^corresponding^analyte-specific^probe^ for^an^ individual^analyte^are^used. 24. The^method^according^to^any^one^of^items^1^to^23,^wherein^the^sample^is^contacted^with^ at^least^two^different^sets^of^signal^oligonucleotides,^wherein^the^signal^oligonucleotides^ RES-PA13-PCT in^each^set^comprise^a^different^signal^element^and^comprise^a^different^connector^element^ (C).^ 25. The^method^according^to^any^one^of^items^1^to^24,^wherein^the^sample^is^contacted^with^ at^least^two^different^sets^of^decoding^oligonucleotides^per^analyte,^ wherein^the^decoding^oligonucleotides^comprised^in^these^different^sets^comprise^ the^ same^ identifier^ connector^ element^ (t)^ comprising^ a^ nucleotide^ sequence^ which^ is^ essentially^complementary^to^at^least^a^section^of^the^unique^identifier^sequence^of^the^ identifier^element^(T)^of^the^corresponding^analyte-specific^probe^set,^and^ wherein^the^decoding^oligonucleotides^of^the^different^sets^per^analyte^differ^in^the^ translator^element^(c)^comprising^a^nucleotide^sequence^allowing^a^specific^hybridization^ of^a^signal^oligonucleotide.^ 26. The^method^according^to^any^one^of^items^1^to^25,^wherein^the^sample^is^contacted^with^ at^least^two^different^sets^of^decoding^oligonucleotides^per^analyte,^ wherein^the^decoding^oligonucleotides^comprised^in^these^different^sets^comprise^ the^ same^ identifier^ connector^ element^ (t)^ comprising^ a^ nucleotide^ sequence^ which^ is^ essentially^complementary^to^at^least^a^section^of^the^unique^identifier^sequence^of^the^ identifier^element^(T)^of^the^corresponding^analyte-specific^probe^set,^and^ wherein^the^decoding^oligonucleotides^of^the^different^sets^per^analyte^differ^in^the^ translator^element^(c)^comprising^a^nucleotide^sequence^allowing^a^specific^hybridization^ of^a^signal^oligonucleotide; wherein^only^one^set^of^decoding^oligonucleotides^per^analyte^is^used^per^cycle,^ and / or^wherein^different^sets^of^decoding^oligonucleotides^are^used^in^different^cycles^in^ combination^with^the^corresponding^set^of^signal^oligonucleotides^in^the^same^cycle. 27. The^method^according^to^of^any^one^of^items^1^to^26,^wherein^the^number^of^different^sets^ of^ decoding^ oligonucleotides^ per^ analyte^ comprising^ different^ translator^ elements^ (c)^ corresponds^ to^ the^ number^ of^ different^ sets^ of^ signal^ oligonucleotides^ comprising^ different^connector^elements^(C).^ RES-PA13-PCT 28. The^ method^ according^ to^ of^ any^ one^ of^ items^ 1^ to^ 27,^ wherein^ all^ sets^ of^ decoding^ oligonucleotides^ for^ the^ different^ analytes^ comprise^ the^ same^ type(s)^ of^ translator^ element(s)^(c). 29. The^method^according^to^any^one^of^items^1^to^28,^wherein^the^different^sets^of^decoding^ oligonucleotides^ may^ be^ comprised^ in^ a^ pre-mixture^ of^ different^ sets^ of^ decoding^ oligonucleotides^or^exist^separately.^ 30. The^method^according^to^any^one^of^items^1^to^29,^wherein^the^different^sets^of^analyte- specific^probes^may^be^ comprised^ in^ a^pre-mixture^of^different^sets^ of^ analyte-specific^ probes^or^exist^separately. 31. The^method^according^to^any^one^of^ items^1^ to^30,^wherein^ the^different^sets^of^signal^ oligonucleotides^ may^ be^ comprised^ in^ a^ pre-mixture^ of^ different^ sets^ of^ signal^ oligonucleotides^or^exist^separately. 32. The^method^according^ to^ any^one^of^ items^1^ to^31,^wherein^ the^ sample^ is^ a^ biological^ sample,^preferably^comprising^biological^tissue,^further^preferably^comprising^biological^ cells^and / or^extracts^and / or^part^of^cells. 33. The^method^according^to^item32,^wherein^the^cell^is^a^prokaryotic^cell^or^a^eukaryotic^cell,^ in^particular^a^mammalian^cell,^in^particular^a^human^cell. 34. The^method^according^to^item 33,^wherein^the^biological^tissue,^biological^cells,^extracts^ and / or^part^of^cells^are^fixed.^ 35. The^method^according^ to^any^one^of^ items^1^ to^34,^wherein^ the^analytes^are^ fixed^ in^a^ permeabilized^sample,^such^as^a^cell-containing^sample. 36. The^method^ according^ to^ any^ one^ of^ items^ 1^ to^ 33,^wherein^ the^ non-bound^ identifier^ elements^(Z)^in^the^premixed^set^of^analyte^specific^probes^are^removed,^in^particular^by^ washing. RES-PA13-PCT 37. The^method^according^to^any^one^of^items^1^to^35,^wherein^after^step^A)^and^before^step^ B)^the^non-bound^analyte-specific^probes^are^removed,^in^particular^by^washing. 38. The^method^according^to^any^one^of^items^1^to^37,^wherein^after^step^C)^and^before^step^ D)^ the^ non-bound^ signal^ oligonucleotides^ and / or^ the^ non-bound^ decoding^ oligonucleotides^are^removed,^in^particular^by^washing. 39. The^method^according^to^any^one^of^items^1^to^38,^wherein^the^analyte^specific^probes^are^ incubated^with^ the^ sample,^ thereby^ allowing^ a^ specific^ binding^ of^ the^ analyte^ specific^ probes^to^the^analytes^to^be^encoded. 40. The^method^according^to^any^one^of^items^1^to^39,^wherein^the^decoding^oligonucleotides^ are^incubated^with^the^sample,^thereby^allowing^a^specific^hybridization^of^the^decoding^ oligonucleotides^to^identifier^elements^(T)^of^the^respective^analyte-specific^probes. 41. The^method^according^to^any^of^items^1 to^40,^wherein^the^binding^element^(Y)^comprise^ an^ amino^ acid^ sequence^ allowing^ a^ specific^ binding^ to^ the^ analyte^ to^ be^ encoded,^ in^ particular^wherein^the^analyte^is^a^protein. 42. The^method^according^to^any^of^items^1^to^41,^wherein^the^binding^element^(Y)^comprises^ moieties^which^are^affinity^moieties^from^affinity^substances^or^affinity^substances^in^their^ entirety^selected^from^the^group^consisting^of^antibodies,^antibody^fragments,^anticalin^ proteins,^ receptor^ ligands,^ enzyme^ substrates,^ lectins,^ cytokines,^ lymphokines,^ interleukins,^angiogenic^or^virulence^ factors,^allergens,^peptidic^allergens,^ recombinant^ allergens,^ allergen-idiotypical^ antibodies,^ autoimmune-provoking^ structures,^ tissue- rejection-inducing^ structures,^ immunoglobulin^ constant^ regions^ and^ combinations^ thereof. 43. The^method^ according^ to^ any^ of^ items^ 1^ to^ 42,^wherein^ the^ binding^ element^ (S)^ is^ an^ antibody^or^an^antibody^fragment^selected^from^the^group^consisting^of^Fab,^scFv;^single^ domain,^or^a^fragment^thereof,^bis^scFv,^Fab2,^Fab3,^minibody,^diabody,^triabody, tetrabody^ and^tandab. RES-PA13-PCT 44. The^method^according^to^any^of^items^1^to^43,^wherein^the^signal^caused^by^the^identifier^ fluorophore^(f)^or^the^signal^element^is^detected^by: (a) Imaging^at^least^a^portion^of^the^sample;^and / or (b) Using^an^optical^imaging^technique;^and / or (c) Using^a^fluorescence^imaging^technique;^and / or (d) Multi-color^fluorescence^imaging^technique;^and / or^ (e) Super-resolution^fluorescence^imaging^technique. 45. The^method^according^ to^any^one^of^ items^1^ to^44,^wherein^ the^ analyte^ is^protein,^ the^ identifier^ element^ (Y)^ is^ an^ antibody^ comprising^ a^ fragment^ crystallizable^ region^ (Fc^ region)^as^the^identifier^element^(T),^the^identifier^element^(Z)^is^a^secondary^antibody,^a^ Fab,^Fab2and / or^a^Fab3^fragment^comprising^an^identifier^oligonucleotide^(o). 46. The^method^according^ to^any^one^of^ items^1^ to^45,^wherein^ the^analyte^ is^protein,^ the^ identifier^ element^ (Y)^ is^ an^ antibody^ comprising^ a^ fragment^ crystallizable^ region^ (Fc^ region)^as^the^identifier^element^(T),^the^identifier^element^(Z)^is^a^secondary^antibody,^a^ Fab,^Fab2 and / or^a^Fab3^fragment^comprising^an^identifier^oligonucleotide^(o),^wherein^the^ sample^is^contacted^with^at^least^one^set^of^decoding^oligonucleotides^per^analyte,^wherein^ in^ each^ set^ of^ decoding^ oligonucleotides^ for^ an^ individual^ analyte^ each^ decoding^ oligonucleotide^comprises: (aa)^an^identifier^connector^element^(t)^comprising^a^nucleotide^sequence^ which^ is^ essentially^ complementary^ to^ at^ least^ a^ section^ of^ the^ identifier^ oligonucleotide^(o)^of^an^identifier^element^(Z)^of^the^corresponding^analyte- specific^probe^set,^and (bb)^a^translator^element^(c)^comprising^a^nucleotide^sequence^allowing^a^ specific^hybridization^of^a^signal^oligonucleotide; wherein^the^decoding^oligonucleotides^of^a^set^for^an^individual^analyte^differ^from^ the^ decoding^ oligonucleotides^ of^ another^ set^ for^ a^ different^ analyte^ in^ the^ first^ connect^ element^ (t);^ and^ contacting^ the^ sample^ with^ at^ least^ a^ set^ of^ signal^ oligonucleotides,^each^signal^oligonucleotide^comprising: (aa)^a^translator^connector^element^(C)^comprising^a^nucleotide^sequence^ which^ is^essentially^complementary^ to^at^ least^a^section^of^ the^nucleotide^ RES-PA13-PCT sequence^ of^ a^ translator^ element^ (c)^ comprised^ in^ a^ decoding^ oligonucleotide,^and (bb)^a^signal^element^and / or^an^additional^sequence^that^can^be^detected^by^ hybridizing^ to^ one^ or^ multiple^ complementary^ oligonucleotides^ that^ are^ labeled. 47. The^ method^ according^ to^ any^ one^ of^ items^ 1^ to^ 46,^ wherein^ the^ premixing^ step^ is^ performed^before^step^(A)^performing^at^least^4^different^complexes^of^individual^Ys and^ individual^Zs,^wherein^Z^ is^coupled^to^a^certain^ identifier^oligonucleotide^(o)^ to^ form^4^ different^combinations^such^as^ - Y1-Z(identfier-oligo1) - Y2-Z(identfier-oligo2) - Y3-Z(identfier-oligo3) - Y4-Z(identfier-oligo4) 48. The^method^according^ to^any^one^of^items^1^to^47,^wherein^ the^sets^of^analyte-specific^ probes^ comprising^ the^ complexes^ between^ the^ binding^ element^ (Y)^ and^ the^ identifier^ element^(Z)^are^stored^before^contacting^ the^sample^with^ the^different^sets^of^analyte- specific^probes.^ 49. The^method^according^to^any^one^of^items^1^to^48,^wherein^the^complexes^between^the^ binding^ element^ (Y)^ and^ the^ identifier^ element^ (Z)^ are^ purified^ and / or^ filtered^ before^ contacting^ the^ sample^ with^ the^ complexes^ comprised^ in^ the^ different^ sets^ of^ analyte- specific^probes.^ 50. The^method^according^to^any^one^of^items^1^to^49,^wherein^the^complexes^between^the^ binding^ element^ (Y)^ and^ the^ identifier^ element^ (Z)^ are^ premixed^ in^ separate^multiple^ tubes. 51. A^ kit^ for^ detecting^ different^ analytes^ in^ a^ sample^ simultaneously,^ wherein^ the^ kit^ comprises^separately^at^least^four^(4)^different^sets^of^analyte-specific^probes^for^encoding^ RES-PA13-PCT of^ at^ least^ 4^ different^ analytes,^ each^ set^ of^ analyte-specific^ probes^ interacting^ with^ a^ different^analyte,^wherein^each^analyte-specific^probe^comprises^complexes^having (aa)^a^binding^element^(Y)^that^specifically^interacts^with^one^of^the^different^ analytes^ to^ be^ encoded,^wherein^ each^ binding^ element^ (Y)^ of^ each^ set^ of^ analyte-specific^probes^comprises^an^identical^identifier^element^(T),^and (bb)^ an^ identifier^ element^ (Z)^ that^ specifically^ interact^ and^ bind^ to^ the^ identifier^ element^ (T)^ of^ the^ binding^ element^ (Y),^ wherein^ the^ identifier^ element^ (Z)^ is^ coupled^ to^ an^ identifier^ fluorophore^ (f)^ or an^ identifier^ oligonucleotide^ (o)^ that^ is^ used^ to^ identify^ the^ identity^ of^ the^ identifier^ element^ (Z),^ wherein^ the^ identifier^ fluorophore^ (f)^ or^ the^ identifier^ oligonucleotide^(o)^of^the^identifier^element^(Z)^in^a^particular^set^of^analyte- specific^ probes^ differ^ from^ the^ identifier^ fluorophore^ (f)^ or^ the^ identifier^ oligonucleotide^(o)^of^ the^ identifier^element^(Z)^of^another^set^of^analyte- specific^probes. 52. The^ kit^ according^ to^ item 51,^wherein^ the^different^ sets^ of^ analyte-specific^probes^ are^ comprised^in^separate^multiple^tubes. 53. A^method^according^to any^of^the^proceeding^items^for^the^use^in^an^application^selected^ from^the^group^consisting^of^dentify^regions^of^interest,^for^image^analysis,^develop^cell^ segmentation^algorithms^using^immunofluorescence^images,^to^aid^the^segmentation^of^ cells^ in^ a^ sample,^ to^ establish^ AI-based^ image^ analysis^ algorithm^ using^ immunofluorescence^ images^ to^ identify^ disorders,^ feature^ extraction^ like^ nucleus^ morphology,^ signal^ homogeneity,^ signal^ intensity^ and / or^ centroids,^ to^ determine^ expression^of^proteins^and^genes^of^interest^via^semi-quantitative^approaches,^diagnosis^ and^prognosis^applications,^determination^of^the^distribution^of^a^target^protein^in^the^of^ health^and^disease^organ,^tissue^and / or^cell,^to^predict^the^prognosis^of^diseases^by^using^ disease-related^prognostic^and / or^predictive^biomarkers,^to^provide^disease^diagnosis^e.g.^ tumor^of^unknow^origin,^neurodegenerative^disorders,^muscle^disorders,^brain^ trauma,^ infectious^ diseases,^ to^ predict^ a^ therapeutic^ response,^ subclassification^ of^ diseases, examine^gene^expression^and^to^study^gene^regulation^in^normal^and^pathological^tissues,^ RES-PA13-PCT morphological^ studies,^ recognition^ of^ tissue^ structures^ and / or^ cell^ type^ classifications,^ multiomics^studies,^quantitative^correlation^and^spatial^colocalization^study^between^ISH^ and^ ICH,^ study^ epigenetic^ modifications^ and^ chromatin^ structures,^ identify^ spatial^ distribution,^ and^ cell-specific^ ISH^ signals^ (Cell^ segmentation) and^ determine^ the^ correlation^ of^ genetic^ aberrations^ and / or^ the^ copy^ number^ variations^ (CNV)^ to^ the^ corresponding^proteins. Experimental^data Cryosection^of^murine^kidney^was^probed^for^nestin.^Rabbit^primary^antibody^against^nestin was^ prelabelled^with^Alexa^Flour^647^conjugated^ to^goat^anti-rabbit^IgG^(upper^panel)^or^goat^anti^ rabbit^Fab^(lower^panel)^with^a^ratio^between^primary^antibody^and^secondary^antibody^of^^1:0.2^ and^1:2^(molecules / molecules),^respectively.^The^prelabelling^was^performed^for^30^minutes^and^ residual^secondary^antibody^or^Fab^not^bound^to^primary^antibody^was^blocked^with^0.1%^v / v^ normal^rabbit^serum^for^5^min.^The^ antibody^complexes^were^ added^to^ tissues^and^ incubated^ overnight.^ Images^ were^ captured^ with^ ZEISS^ Celldiscoverer^ 7^ using^ a^ Apochromat^ 5X / 0.35^ objective^and^2X^Tubelens.^(Exposure^time:^1^second^and^0.1^second^for^Alexa^Flour^647^and^DAPI,^ respectively).^The^results^are^shown^in^Figure^3.^^^^^^^^^ Cyrosection^ of^ murine^ kidney^ were^ probed^ for^ Nestin.^ Detection^ of^ nestin^ was^ done^ by^ precomplexed^primary^antibody^(Abcam^ab176571)^with^oligos^conjugated^secondary^antibody^ followed^ by^ complementary^ oligos^ conjugated^ to^ fluorophores^ (A).^ The^ localization^ of^ the^ primary-secondary^antibody^complexes^was^confirmed^by^probing^the^tissue^with^fluorophores^ conjugated^antibody^complemented^to^the^oligo^conjugated^secondary^antibody^(B).^^^The^results^ are^shown^in^Figure^4.^^^^^^^^^

Claims

RES-PA13-PCT Claims 1. A^method^for^detecting^different^analytes^in^a^sample^simultaneously^comprising the^steps^ of:^ (A) contacting^the^sample^with^at^least^four^(4)^different^sets^of^analyte-specific^probes^for^ detecting^of^at^ least^4 different^analytes,^each^set^ of^analyte-specific^probes^ interacting^ with^a^different^analyte,^wherein^each^analyte-specific^probe^is^a^complex comprising (aa)^a^binding element (Y) that^specifically^interacts^with^one^of^the^different^ analytes^ to^ be^ encoded,^wherein^ each^ binding^ element (Y)^ of^ each^ set^ of^ analyte-specific^probes^comprises^an^identical^identifier^element^(T),^and (bb)^ an^ identifier^ element^ (Z) that^ specifically^ interact^ and^ bind^ to^ the^ identifier^ element^ (T)^ of^ the^ binding^ element^ (Y),^ wherein^ the^ identifier^ element^ (Z)^ is^ coupled^ to^ an^ identifier^ fluorophore^ (f)^ or an identifier^ oligonucleotide^ (o)^ that^ is^ used^ to^ identify^ the^ identity^ of^ the^ identifier^ element^ (Z),^ wherein^ the^ identifier^ fluorophore^ (f)^ or^ the^ identifier^ oligonucleotide^(o)^of^the^identifier^element^(Z)^in^a^particular^set^of^analyte- specific^ probes^ differ^ from^ the^ identifier^ fluorophore^ (f)^ or^ the^ identifier^ oligonucleotide^(o)^of^ the^ identifier^element^(Z)^of^another^set^of^analyte- specific^probes, wherein^the^binding^element^(Y)^and^the^identifier^element^(Z)^are^premixed^for each^set^of^analyte-specific^probes in^a^separate^reaction^tube^to^form^complexes^between^ the^binding^element^(Y)^and^the^identifier^element^(Z)^before^contacting^the^sample^with^ the^different^sets^of^analyte-specific^probes,^wherein^the^identifier^element^(Z)^interacts and^binds to^the^identical^identifier^element^(T)^of^the^binding^element^(Y), (B) detecting^the^signal^caused^by^the^identifier^fluorophore^(f)^coupled^to^the^identifier^ element^(Z);^or (C)^ i)^ contacting^ the^ sample^ with^ different^ set^ of^ signal^ oligonucleotides,^ each^ signal^ oligonucleotide^comprising: (aa)^a^translator^connector^element^(C)^comprising^a^nucleotide^sequence^ which^ is^essentially^complementary^ to^at^ least^a^section^of^ the^nucleotide^ 1RES-PA13-PCT sequence^ of^ an^ identifier^ oligonucleotide^ (o)^ comprised^ in^ an identifier^ element^(Z),^and (bb)^a^signal^element and / or^an^additional^sequence^that^can^be^detected^by^ hybridizing to^ one^ or^ multiple^ complementary^ oligonucleotides^ that^ are^ labeled or ii)^contacting^the^sample^with^at^least^one^set^of^decoding^oligonucleotides^per^analyte,^ wherein^in^each^set^of^decoding^oligonucleotides^for^an^individual^analyte^each^decoding^ oligonucleotide^comprises: (aa)^an^identifier^connector^element^(t)^comprising^a^nucleotide^sequence^ which^ is^ essentially^ complementary^ to^ at^ least^ a^ section^ of^ the^ identifier^ oligonucleotide^(o)^of^an identifier^element^(Z) of^the^corresponding^analyte- specific probe^set,^and (bb)^a^translator^element^(c)^comprising^a^nucleotide^sequence^allowing^a^ specific^hybridization^of^a^signal^oligonucleotide; wherein^the^decoding^oligonucleotides^of^a^set^for^an^individual^analyte^differ^from^ the^ decoding^ oligonucleotides of^ another^ set^ for^ a^ different^ analyte^ in^ the^ first^ connect^ element^ (t); and^ contacting^ the^ sample^ with^ at^ least^ a^ set^ of^ signal^ oligonucleotides,^each^signal^oligonucleotide^comprising: (aa)^a^translator^connector^element^(C)^comprising^a^nucleotide^sequence^ which^ is^essentially^complementary^ to^at^ least^a^section^of^ the^nucleotide^ sequence^ of^ a^ translator^ element^ (c)^ comprised^ in^ a^ decoding^ oligonucleotide,^and (bb)^a^signal^element^and / or^an^additional^sequence^that^can^be^detected^by^ hybridizing^ to^ one or^ multiple^ complementary^ oligonucleotides^ that^ are^ labeled; (D)Detecting^the^signal^caused^by^the^signal^element^of^section^C)^i)^and / or^C)^ii);^and^ optionally (E) Selectively^ removing^of^ the^ signal^ oligonucleotides^of^ section^C)^ i)^ or^ the^decoding^ oligonucleotides^ and^ signal^ oligonucleotides^ of^ of^ section^ C)^ ii)^ from^ the^ sample,^ thereby^essentially^maintaining^the^specific^binding^of^the^analyte-specific^probes^to^ the^analytes^to^be^encoded;^and^optionally 2RES-PA13-PCT (F) Performing^at^least^three^(3)^further^cycles^comprising^steps^C)^to^E)^to^generate^an^ encoding^scheme^with^a^code^word^per^analyte,^wherein^in^particular^the^last^cycle^ may^stop^with^step^(D).

2. The^method^according^to^claim^1,^wherein^the^sample^is^a^biological^sample,^preferably^ comprising^ biological^ tissue from^ human,^ animals,^ or^ plants,^ further^ preferably^ comprising^biological^cells^and / or^extracts^and / or^part^of^cells.

3. The^method^to^any^one^of^claims^1^to^2,^wherein^the^cell^is^a^prokaryotic^cell or^a^eukaryotic^ cell,^an^animal^cell,^a^plant^cell,^ in^particular^a^cell from^vertebrate^cells,^ in^particular^a^ mammalian^cell,^in^particular^a^human^cell.

4. The^method^according^to^claim^2,^wherein^the^biological^tissues,^biological^cells,^extracts^ and / or^part^of^cells^are^fixed.^ 5. The method^according^ to^any^one^of^ claims^1^ to^4,^wherein^ the^analytes^are^ fixed^ in^a^ permeabilized^sample,^such^as^a^cell-containing^sample.

6. The^method^according^to^any^one^of^claims^1^to^5,^wherein^the^analyte^is^a^nucleic^acid,^ preferably^DNA,^PNA^or^RNA, in^particular^mRNA.

7. The^method^ according^ to^ any^ one^ of^ claims^ 1^ to^ 5,^ wherein^ the^ analyte^ is^ a^ peptide,^ polypeptide^or^a^protein.

8. The^method^according^to^any^one^of^claims^1^to^7,^wherein^the^binding^element^(Y)^is^a^ protein^and / or^a^nucleic^acid^that^specifically^recognizes^and^binds^to^the^analyte.

9. The^method^according^to^any^one^of^claims^1^to^8,^wherein^the^binding^element^(Y)^is^an^ antibody,^an^antibody fragment,^an^aptamer^or^a^nucleic^acid^that^specifically^interacts and^ binds to^the^analyte, wherein^in^particular^the^antibody^fragment^is^selected^from^the^group^ consisting^of^a^Fab,^scFv,^single^domain,^bis^scFv,^Fab2,^Fab3,^minibody,^diabody,^triplebody,^ tetrabody^and^tandab.

10. The^method^according^to^any^one^of^claims^1^to^9,^wherein^the^identifier^element^(Z)^is^an^ antibody,^an^antibody^fragment,^an^aptamer^or^a^nucleic^acid^that^specifically^interacts^and^ binds^to^the^identifier^element^(Z), wherein^in^particular^the^antibody^fragment^is^selected^ 3RES-PA13-PCT from^ the^ group^ consisting^ of^ a^ Fab,^ scFv, single^ domain,^ bis^ scFv,^ Fab2,^ Fab3,^ minibody,^ diabody,^triplebody,^tetrabody^and^tandab.

11. The^method^according^to^any^one^of^claims^1^to^10,^wherein^the^identifier^element^(Z)^is^ an^antibody^fragment selected^from^the^group^consisting^of^a^Fab,^scFv,^single^domain,^bis^ scFv,^Fab2,^Fab3,^minibody,^diabody,^triplebody,^tetrabody^and^tandab, wherein^^in^particular^ the^antibody^fragment^is^a^Fab.

12. The^method^ according^ to^ any^ one^ of^ claims^ 1^ to^ 11,^wherein^ the^ analyte^ is^ a^ protein^ comprised^in^a^biological^sample,^the^binding^element^(Y)^is^an^antibody^or^an^antibody^ fragment^comprising^a^(Fc)^region and^the^identifier^element^(Z)^is a^secondary^antibody,^ a^Fab,^Fab2 and / or^a^Fab3^fragment.

13. The^method^according^to^any^one^of^claims^1^to^12,^wherein^after^step A)^and^before^step^ B)^ the^ identifier^ elements (Z) are^ blocked^ to^ prevent^ cross^ reactivity,^ in^ particular^ by^ mixing^with^the^binding^element^specific^for^the^identifier^element (Z).^ 14. The^method^according^to^any^one^of^claims^1^to^3 wherein^after^step^A)^and^before^step B)^ the^non-bound^analyte-specific^probes^are^removed,^in^particular^by^washing.

15. The^method^according^to^any^one^of^claims^1^to^14,^wherein^all^steps^are^automated,^in^ particular^by^using^a^robotic^system.

16. The^method^according^to^any^one^of claims^1^to^15,^wherein^all^steps^are^performed^in^a^ fluidic^system.

17. The^method^according^to^any^one^of^claims^1^to^16,^wherein^each^analyte^is^associated^ with^a^specific^code^word,^wherein^said^code^word^comprise^a^number^of^positions,^and^ wherein^each^position^corresponds^to^one^cycle^resulting^in^a^plurality^of^distinguishable^ encoding^schemes^with^the^plurality^of^code^words.

18. The^method^according^ to^ any^one^ of^ claims^1^ to^17,^wherein^ said^ encoding^ scheme^ is^ predetermined^and^allocated^to^the^analyte^to^be^encoded.

19. The^method^according^to^any^one^of^claims^1^to^18,^wherein^the^code^words^obtained^for^ the^ individual^ analytes^ in^ the^ performed^ cycles^ comprise^ the^ detected^ signals^ and^ additionally^at^least^one^element^corresponding^to^no^detected^signal. 4RES-PA13-PCT 20. The^method^according^to^any^one^of^claims^1^to^19,^wherein^no^signal^is^detected^for^at^ least^one^analyte^within^at^least^one^cycle.

21. The^method^according^to^any^one^of^claims^1^to^20,^wherein^for^at^least^for^one^individual^ analyte^a^position^of^the^code word^is^zero^(0).

22. The^method^according^to^any^one^of^claims^1^to^21,^wherein^the^code^word^zero^(0)^is^ generated^by^using^no^decoding^oligonucleotides^having^an^identifier^connector^element^ (t)^ comprising^ a^nucleotide^ sequence^which^ is^ essentially^ complementary^ to^ at^ least^ a^ section^of^the^unique^identifier^sequence^of^the^identifier^element^(T)^of^a^corresponding^ analyte-specific^probe^for^an^individual^analyte.

23. The^method^according^to^any^one^of^claims^1^to^22,^wherein^if^at^least^for^one^individual^ analyte^a^position^of^the^code^word^is^zero^(0)^in^this^cycle^no^corresponding^decoding^ oligonucleotides^ having^ an^ identifier^ connector^ element^ (t)^ comprising^ a^ nucleotide^ sequence^which^is^essentially^complementary^to^at^least^a^section^of^the^unique^identifier^ sequence^of^ the^ identifier^element^(T)^of^a^corresponding^analyte-specific^probe^ for^an^ individual^analyte^are^used.

24. The^method^according^to^any^one^of^claims^1^to^23,^wherein^the^sample^is^contacted^with^ at^least^two^different^sets^of^signal^oligonucleotides,^wherein^the^signal^oligonucleotides^ in^each^set^comprise^a^different^signal^element^and^comprise^a^different^connector^element^ (C).^ 25. The^method^according^to^any^one^of^claims^12^to^24,^wherein^the^sample^is^contacted^with^ at^least^two^different^sets^of^decoding^oligonucleotides^per^analyte,^ wherein^the^decoding^oligonucleotides^comprised^in^these^different^sets^comprise^ the^ same^ identifier^ connector^ element^ (t)^ comprising^ a^ nucleotide^ sequence^ which^ is^ essentially^complementary^to^at^least^a^section^of^the^unique^identifier^sequence^of^the^ identifier^element^(T)^of^the^corresponding^analyte-specific^probe^set,^and^ wherein^the^decoding^oligonucleotides^of^the^different^sets^per^analyte^differ^in^the^ translator^element^(c)^comprising^a^nucleotide^sequence^allowing^a^specific^hybridization^ of^a^signal^oligonucleotide.^ 26. The^method^according^to^any^one^of^claims^1 to^25,^wherein^the^sample^is^contacted^with^ at^least^two^different^sets^of^decoding^oligonucleotides^per^analyte,^ 5RES-PA13-PCT wherein^the^decoding^oligonucleotides^comprised^in^these^different^sets^comprise^ the^ same^ identifier^ connector^ element^ (t)^ comprising^ a^ nucleotide sequence^ which^ is^ essentially^complementary^to^at^least^a^section^of^the^unique^identifier^sequence^of^the^ identifier^element^(T)^of^the^corresponding^analyte-specific^probe^set,^and^ wherein^the^decoding^oligonucleotides^of^the^different^sets^per^analyte differ^in^the^ translator^element^(c)^comprising^a^nucleotide^sequence^allowing^a^specific^hybridization^ of^a^signal^oligonucleotide; wherein^only^one^set^of^decoding^oligonucleotides^per^analyte^is^used^per^cycle,^ and / or^wherein^different^sets^of^decoding oligonucleotides^are^used^in^different^cycles^in^ combination^with^the^corresponding^set^of^signal^oligonucleotides^in^the^same^cycle.

27. The^method^according^to^of^any^one^of^claims^1 to^26,^wherein^the^number^of^different^sets^ of^ decoding^ oligonucleotides^ per^ analyte^ comprising^ different^ translator^ elements^ (c)^ corresponds^ to^ the^ number^ of^ different^ sets^ of^ signal^ oligonucleotides^ comprising^ different^connector^elements^(C).^ 28. The^ method^ according^ to^ of^ any^ one^ of^ claims^ 1 to^ 27,^ wherein^ all^ sets^ of^ decoding^ oligonucleotides^ for^ the^ different^ analytes^ comprise^ the^ same^ type(s)^ of^ translator^ element(s)^(c).

29. The^method^according^to^any^one^of^claims^1 to^28,^wherein^the^different^sets^of^decoding^ oligonucleotides^ may^ be^ comprised^ in^ a^ pre-mixture^ of^ different sets^ of^ decoding^ oligonucleotides^or^exist^separately.^ 30. The^method^according^to^any^one^of^claims^1 to^29,^wherein^the^different^sets^of^analyte- specific^probes^may^be^ comprised^ in^ a^pre-mixture^of^different^sets^ of^ analyte-specific^ probes^or^exist^separately.

31. The^method^according^to^any^one^of^claims^1 to^30,^wherein^the^different^sets^of^signal^ oligonucleotides^ may^ be^ comprised^ in^ a^ pre-mixture^ of^ different^ sets^ of^ signal^ oligonucleotides^or^exist^separately.

32. The^method^according^ to^any^one^of^claims^1 to^31,^wherein^ the^sample^ is^a^biological^ sample,^preferably^comprising^biological^tissue,^further^preferably^comprising^biological^ cells^and / or^extracts^and / or^part^of^cells. 6RES-PA13-PCT 33. The^method^according^to^claim^32,^wherein^the^cell^is^a^prokaryotic^cell or^a^eukaryotic^ cell,^in^particular^a^mammalian^cell,^in^particular^a^human^cell.

34. The^method^according^to^claim^32,^wherein^the^biological^tissue,^biological^cells,^extracts^ and / or^part^of^cells^are^fixed.^ 35. The^method^according^to^any^one^of^claims^1 to 34,^wherein^the^analytes^are^fixed^in^a^ permeabilized^sample,^such^as^a^cell-containing^sample.

36. The^method^according^ to^ any^one^of^ claims^1^ to^35,^wherein^ the^non-bound^ identifier^ elements^(Z)^in^the^premixed^set^of^analyte^specific^probes^are^removed,^in^particular^by^ washing.

37. The^method^according^to^any^one^of^claims^1 to^36,^wherein^after^step^A)^and^before^step^ B)^the^non-bound^analyte-specific^probes^are^removed,^in^particular^by^washing.

38. The^method^according^to^any^one^of^claims^1 to^37,^wherein^after^step^C)^and^before^step^ D)^ the^ non-bound^ signal^ oligonucleotides and / or^ the^ non-bound^ decoding^ oligonucleotides are^removed,^in^particular^by^washing.

39. The^method^according^to^any^one^of^claims^1 to^38,^wherein^the^analyte^specific^probes^ are^incubated^with^the^sample,^thereby^allowing^a^specific^binding^of^the^analyte^specific^ probes^to^the^analytes^to^be^encoded.

40. The^method^according^to^any^one^of^claims^1 to^39,^wherein^the^decoding^oligonucleotides^ are^incubated^with^the^sample,^thereby^allowing^a^specific^hybridization^of^the^decoding^ oligonucleotides^to^identifier^elements^(T)^of^the^respective^analyte-specific^probes.

41. The^method^according^to^any^of^claims^1 to^40,^wherein^the^binding^element^(Y)^comprise^ an^ amino^ acid^ sequence^ allowing^ a^ specific^ binding^ to^ the^ analyte^ to^ be^ encoded,^ in^ particular^wherein^the^analyte^is^a^protein.

42. The^method^according^to^any^of^claims^1 to^41,^wherein^the^binding^element^(Y)^comprises^ moieties^which^are^affinity^moieties^from^affinity^substances^or^affinity^substances^in^their^ entirety^selected^from^the^group^consisting^of^antibodies,^antibody^fragments,^anticalin^ proteins,^ receptor^ ligands,^ enzyme^ substrates,^ lectins,^ cytokines,^ lymphokines,^ interleukins,^angiogenic^or^virulence^ factors,^allergens,^peptidic^allergens,^ recombinant^ 7RES-PA13-PCT allergens,^ allergen-idiotypical^ antibodies,^ autoimmune-provoking^ structures,^ tissue- rejection-inducing^ structures,^ immunoglobulin^ constant^ regions^ and^ combinations^ thereof.

43. The^method^according^ to^any^of^claims^1 to^42,^wherein^ the^binding^element^(S)^ is^an^ antibody^or^an^antibody^fragment^selected^from^the^group^consisting^of^Fab,^scFv;^single^ domain,^or^a^fragment^thereof,^bis^scFv,^Fab2,^Fab3,^minibody,^diabody,^triabody,^tetrabody^ and^tandab.

44. The^method^according^to^any^of^claims^1 to^43,^wherein^the^signal^caused^by^the^identifier^ fluorophore^(f)^or^the^signal^element is^detected by: (a) Imaging^at^least^a^portion^of^the^sample;^and / or (b) Using^an^optical^imaging^technique;^and / or (c) Using^a^fluorescence^imaging^technique;^and / or (d) Multi-color^fluorescence^imaging^technique;^and / or^ (e) Super-resolution^fluorescence^imaging^technique.

45. The^method^according^to^any^one^of^claims^1^to^44,^wherein^the^analyte is^protein,^the^ identifier^ element^ (Y)^ is^ an^ antibody^ comprising^ a^ fragment^ crystallizable^ region^ (Fc^ region)^as^the^identifier^element^(T),^the^identifier^element^(Z)^is^a^secondary^antibody,^a^ Fab,^Fab2and / or^a^Fab3^fragment^comprising^an^identifier^oligonucleotide^(o).

46. The^method^according^to^any^one^of^claims^1^to^45,^wherein^the^analyte^is^protein,^the^ identifier^ element^ (Y)^ is^ an^ antibody^ comprising^ a^ fragment^ crystallizable^ region^ (Fc^ region)^as^the^identifier^element^(T),^the^identifier^element^(Z)^is^a^secondary^antibody,^a^ Fab,^Fab2 and / or^a^Fab3^fragment^comprising^an^identifier^oligonucleotide^(o),^wherein^the^ sample^is^contacted^with^at^least^one^set^of^decoding^oligonucleotides^per^analyte,^wherein^ in^ each^ set^ of^ decoding^ oligonucleotides^ for^ an^ individual^ analyte^ each^ decoding^ oligonucleotide^comprises: (aa) an^identifier^connector^element^(t)^comprising^a^nucleotide^sequence^ which^ is^ essentially^ complementary^ to^ at^ least^ a^ section^ of^ the^ identifier^ oligonucleotide^(o)^of^an^identifier^element^(Z)^of^the^corresponding^analyte- specific^probe^set,^and (bb)^a^translator^element^(c)^comprising^a^nucleotide^sequence^allowing^a^ specific^hybridization^of^a^signal^oligonucleotide; 8RES-PA13-PCT wherein^the^decoding^oligonucleotides^of^a^set^for^an^individual^analyte^differ^from^ the^ decoding^ oligonucleotides^ of^ another^ set^ for^ a^ different^ analyte^ in^ the^ first^ connect^ element^ (t);^ and^ contacting^ the^ sample^ with^ at^ least^ a^ set^ of^ signal^ oligonucleotides,^each^signal^oligonucleotide^comprising: (aa)^a^translator^connector^element^(C)^comprising^a^nucleotide^sequence^ which^ is^essentially complementary^ to^at^ least^a^section^of^ the^nucleotide^ sequence^ of^ a^ translator^ element^ (c)^ comprised^ in^ a^ decoding^ oligonucleotide,^and (bb)^a^signal^element^and / or^an^additional^sequence^that^can^be^detected^by^ hybridizing^ to^ one^ or^ multiple^ complementary^ oligonucleotides^ that^ are^ labeled.

47. The^ method^ according^ to^ any^ one^ of^ claims^ 1^ to^ 46,^ wherein^ the^ premixing^ step^ is^ performed^before^step^(A)^performing^at^least^4^different^complexes^of^individual^Ys^and^ individual^Zs,^wherein^Z^ is^coupled^ to^a^certain identifier^oligonucleotide^(o)^ to^ form^4^ different^combinations^such^as^ - Y1-Z(identfier-oligo1) - Y2-Z(identfier-oligo2) - Y3-Z(identfier-oligo3) - Y4-Z(identfier-oligo4) 48. The^method^according^to^any^one^of^claims^1^to^47,^wherein^the^sets^of^analyte-specific^ probes^ comprising^ the complexes^ between^ the^ binding^ element^ (Y)^ and^ the^ identifier^ element^(Z)^are^stored^before^contacting^ the^sample^with^ the^different^sets^of^analyte- specific^probes.^ 49. The^method^according^to^any^one^of^claims^1^to^48,^wherein the^complexes^between^the^ binding^ element^ (Y)^ and^ the^ identifier^ element^ (Z)^ are^ purified^ and / or^ filtered^ before^ contacting^ the^ sample^ with^ the^ complexes^ comprised^ in^ the^ different^ sets^ of^ analyte- specific^probes.^ 50. The^method^according^to^any^one^of^claims^1^to^49,^wherein^the^complexes^between^the^ binding^ element^ (Y)^ and^ the^ identifier^ element^ (Z)^ are^ premixed^ in^ separate^multiple^ tubes.

51. The^method^according^to^any^one^of^claims^1^to^50,^wherein^the^sample^is^contacted^with^ at^ least^ four^ (4)^ additional^ sets^ of^ analyte^ specific^probes^ for^detecting^of^ at^ least^ four 9RES-PA13-PCT additional^analytes,^wherein^each^set^of^the^additional^analyte-specific^probes^comprises^ an^identical^identifier^element^(T) that^is^not^identical^with^the^identical^identifier^element^ (T)^comprised^in^the^complexes^of^the^first^four^sets^of^analyte-specific^probes.

52. A^ kit^ for^ detecting^ different^ analytes^ in^ a^ sample^ simultaneously,^ wherein^ the^ kit comprises^separately^at^least four^(4)^different^sets^of^analyte-specific^probes^for^encoding^ of^ at^ least^ 4^ different^ analytes,^ each^ set^ of^ analyte-specific^ probes^ interacting^ with^ a^ different^analyte,^wherein^each^analyte-specific^probe^comprises^complexes^having (aa)^a^binding^element^(Y)^that^specifically^interacts^with^one^of^the^different^ analytes^ to^ be^ encoded,^wherein^ each^ binding^ element^ (Y)^ of^ each^ set^ of^ analyte-specific^probes^comprises^an^identical^identifier^element^(T),^and (bb)^ an^ identifier^ element^ (Z)^ that^ specifically^ interact^ and^ bind^ to^ the^ identifier^ element^ (T)^ of^ the^ binding^ element^ (Y),^ wherein^ the^ identifier^ element^ (Z)^ is^ coupled^ to^ an^ identifier^ fluorophore^ (f)^ or an^ identifier^ oligonucleotide^ (o)^ that^ is^ used^ to^ identify^ the^ identity^ of^ the^ identifier^ element^ (Z),^ wherein^ the^ identifier^ fluorophore^ (f)^ or^ the^ identifier^ oligonucleotide^(o)^of^the^identifier^element^(Z)^in^a^particular^set^of^analyte- specific^ probes^ differ^ from^ the^ identifier^ fluorophore^ (f)^ or^ the^ identifier^ oligonucleotide^(o)^of^ the^ identifier^element^(Z)^of^another^set^of^analyte- specific^probes.

53. The^kit^according^ to^claim^52,^wherein^ the^different^sets^of^analyte-specific^probes are^ comprised^in^separate^multiple^tubes.

54. A^method^according^to^any^of^the^proceeding^claims^for^the^use^in^an^application^selected^ from^the^group^consisting^of^identify^regions^of^interest,^for^image^analysis,^develop^cell^ segmentation^algorithms^using^immunofluorescence^images,^to^aid^the^segmentation^of^ cells^ in^ a^ sample,^ to^ establish^ AI-based^ image^ analysis^ algorithm^ using^ immunofluorescence^ images^ to^ identify^ disorders,^ feature^ extraction^ like^ nucleus^ morphology,^ signal^ homogeneity,^ signal^ intensity^ and / or^ centroids,^ to^ determine^ expression^of proteins^and^genes^of^interest^via^semi-quantitative^approaches,^diagnosis^ and^prognosis^applications,^determination^of^the^distribution^of^a^target^protein^in^the^of^ health^and^disease^organ,^tissue^and / or^cell,^to^predict^the^prognosis^of^diseases^by^using^ disease-related^prognostic^and / or^predictive^biomarkers,^to^provide^disease^diagnosis^e.g.^ tumor^of^unknow^origin,^neurodegenerative^disorders,^muscle^disorders,^brain^trauma,^ 10RES-PA13-PCT infectious^ diseases,^ to^ predict^ a^ therapeutic^ response,^ subclassification^ of diseases,^ examine^gene^expression^and^to^study^gene^regulation^in^normal^and^pathological^tissues,^ morphological^ studies,^ recognition^ of^ tissue^ structures^ and / or^ cell^ type^ classifications,^ multiomics^studies,^quantitative^correlation^and^spatial^colocalization^study^between^ISH^ and^ ICH,^ study^ epigenetic^ modifications^ and^ chromatin^ structures,^ identify^ spatial^ distribution,^ and^ cell-specific^ ISH^ signals^ (Cell^ segmentation)^ and^ determine^ the^ correlation^ of^ genetic^ aberrations^ and / or^ the^ copy^ number^ variations^ (CNV)^ to^ the^ corresponding^proteins. 11

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