A method of multicolor immunofluorescence staining and imaging

Abstract

The application provides application of a compound shown in formula (III) in multicolor immunofluorescence staining. The method can be used for marking a plurality of different fluorescent dyes on a plurality of antigens in a sample, and then the fluorescent dyes in the last round of staining can be eluted through an elution process, so that a new round of multicolor immunofluorescence marking can be started. The marked sample can be used for imaging by instruments and equipment such as a fluorescence microscope or a laser confocal microscope, and after software synthesis, the sample marked by a plurality of different colors can be displayed. The colors are clear, bright and do not bleed. The application not only retains the sensitivity and staining intensity of the TSA fluorescent staining method, but also overcomes the limitation of the number of multicolor fluorescent markers.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunoassay, in particular to a multi-color immunofluorescence staining method and imaging method. BACKGROUND

[0002] Immunofluorescence staining methods can be roughly divided into two categories according to technical principles, traditional immunofluorescence staining method and tyramide signal amplification (TSA) based immunofluorescence staining method.

[0003] Traditional immunofluorescence staining technology is based on the principle of antigen-antibody reaction. First, known antigens or antibodies are labeled with fluorescent substances (such as fluorescein) to make fluorescently labeled antibodies (or antigens). Then, the fluorescently labeled antibodies are used as molecular probes to detect the corresponding antigens (or antibodies) in cells or tissues. Finally, the antigen-antibody complex formed in the cells or tissues contains fluorescent substances (such as fluorescein). The sample is observed using a fluorescence microscope. The fluorescent substance emits bright fluorescence (such as fluorescein emits bright green fluorescence) under excitation light. The cells or tissues where the fluorescence is located can be seen, thereby determining the properties, localization of antigens or antibodies, and measuring the content of target antigens (or antibodies) using quantitative techniques. Multi-color immunofluorescence labeling can be achieved by using antibodies labeled with different fluorescent substances for staining. The disadvantages of traditional immunofluorescence staining technology for multi-color immunofluorescence labeling are as follows: 1. Multiple fluorescently labeled antibodies need to be prepared. The activity of fluorescently labeled antibodies may have an adverse effect on the antibodies, reducing the sensitivity of antibody detection. 2. If the secondary antibody is labeled with a fluorescent substance, additional attention needs to be paid to the species pairing relationship between the primary antibody and the fluorescently labeled secondary antibody during multi-color immunofluorescence labeling. Otherwise, cross-reactions may occur, resulting in false positive experimental results. 3. The staining intensity is low, and false negative results may occur when detecting low expression of the target to be detected.

[0004] Tyramide signal amplification technology (TSA) is based on the principle of signal molecule precipitation (CARD) on the basis of traditional immunohistochemical staining, in the presence of H2O2, horseradish peroxidase (HRP) labeled on the antibody converts fluorescently labeled substrate tyramide (T) into a transiently active intermediate state (T*), and then the activated substrate molecules rapidly and stably covalently bind to the electron-rich region (tyrosine residues) of the adjacent protein molecules; Because the adjacent proteins (antigens, HRP, etc.) all contain a large number of tyrosine binding sites, a large amount of signal will be enriched, so the signal will be effectively amplified. After multiple rounds of staining of different types of fluorescently labeled substrate tyramide, multi-color immunofluorescence labeling can be achieved on one sample. The advantage of applying the principle of TSA technology to realize multi-color fluorescent staining is that the fluorescent signal is covalently bound to the target sample and is not easily lost, and after the HRP amplification process, the signal is strong. The disadvantage is that due to the cross-reaction of the fluorescent signal, when the number of different fluorescent dyes used for staining is large, color mixing may occur. The number of multi-color labeling is limited, and generally only 4-7 colors of fluorescent staining can be performed on one sample. SUMMARY

[0005] Therefore, the technical problem to be solved by the present application is to overcome the defects in the prior art that different fluorescent dyes need to be used for multi-color staining, there is a risk of mutual color mixing interference between different fluorescent dyes, and only a limited number of target objects can be labeled on the same sample, thereby providing an experimental technique for realizing multi-color immunofluorescence labeling on one sample by using a single fluorescent dye or several fluorescent dyes through cyclic staining.

[0006] The present application provides the use of a compound represented by the following formula (III) in multi-color immunofluorescence staining; or, the use of a compound represented by the following formula (III) in the preparation of a multi-color immunofluorescence staining product;

[0007]

[0008] wherein R5 is a fluorescent dye.

[0009] Optionally, R1 and R2 in formula (III) are each selected from any one of hydrogen, methyl, ethyl and hydroxyl;

[0010] R3 is any chemical group connecting the disulfide bond and the benzene ring;

[0011] R4 is any chemical group connecting the disulfide bond and R5.

[0012] Optionally, R3 is selected from -CH2-CH2-, -C=C-, an amide bond, an ester bond;

[0013] R4 is selected from -CH2-CH2-, -C=C-, an amide bond, an ester bond.

[0014] Optionally, R5 is any one selected from the group consisting of coumarin, cyanine, fluorescein, rhodamine, Bodipy, and NBD amine, and other code series dyes;

[0015] Optionally, the coumarin dye is any one of coumarin and its derivatives;

[0016] Optionally, the cyanine dye is any one of CY2, CY3, CY5, CY5.5, CY7 and CY7.5;

[0017] Optionally, the fluorescein dye is any one of FAM, HEX, JOE and TET series dyes;

[0018] Optionally, the rhodamine dye is any one of rhodamine B, rhodamine 6G and rhodamine 101 series dyes;

[0019] Optionally, the Bodipy dye is any one of boron-difluoride dipyrrin fluorescent dye and its derivatives;

[0020] Optionally, the NBD amine dye is any one of benzofurazan compound and its derivatives;

[0021] Optionally, the other code series dye is any one of Alexa Fluor Dyes, mFlour Dyes, iFluor Dyes, CF Dyes series fluorescent dyes and its derivatives;

[0022] The compound.

[0023] A multi-color immunofluorescence staining method, comprising the following steps: after the target protein is stained by the compound, the disulfide bond in the compound is broken by β-mercaptoethanol, DTT or TECP, and reduced to -SH.

[0024] A multi-color immunofluorescence staining method, comprising the following steps:

[0025] S1: first round of staining

[0026] S11: first staining of the first round;

[0027] S111: A protein specific antibody incubates the sample to obtain sample 111;

[0028] S112: Anti-A protein specific antibody incubates sample 111 to obtain sample 112;

[0029] S113: The A protein of sample 112 is stained by the compound represented by formula (III);

[0030] S12: Second staining of the first round

[0031] S121: Incubate the sample with B protein specific antibody to obtain sample 121;

[0032] S122: Incubate sample 121 with anti-B protein specific antibody to obtain sample 122;

[0033] S123: Stain sample 122 with compound of formula (III) for B protein;

[0034] S13: Capture images;

[0035] S14: Break the disulfide bond in the compound with β-mercaptoethanol, DTT or TECP to reduce to -SH;

[0036] S2: Second round of staining

[0037] S21: First staining of the second round;

[0038] S211: Incubate the sample with C protein specific antibody to obtain sample 211;

[0039] S212: Incubate sample 211 with anti-C protein specific antibody to obtain sample 212;

[0040] S213: Stain sample 212 with compound of formula (III) for C protein;

[0041] S22: Second staining of the second round;

[0042] S221: Incubate the sample with D protein specific antibody to obtain sample 21;

[0043] S222: Incubate sample 211 with anti-D protein specific antibody to obtain sample 212;

[0044] S223: Stain sample 212 with compound of formula (III) for D protein;

[0045] S23: Capture images;

[0046] S24: Break the disulfide bond in the compound with β-mercaptoethanol, DTT or TECP to reduce to -SH;

[0047] By analogy, perform the third to Nth round of staining; N is an integer ≥ 3 and ≤ 10;

[0048] The Nth round of staining comprises any one of:

[0049] 3) first staining of the Nth round and second staining of the Nth round;

[0050] 4) the 1st staining of the Nth round;

[0051] The color of the fluorescent dye of the compound shown in formula (III) used in the 2nd staining in any one of the 1st to Nth round of staining is different.

[0052] Optionally, after the step of "breaking the disulfide bond in the compound into -SH by using beta-mercaptoethanol, DTT or TECP" in any one of the 1st to Nth round of staining, the method further comprises the step of adding a blocking solution dropwise.

[0053] Optionally, when the sample is a paraffin tissue section, the method further comprises the step of deparaffinizing and hydrating the paraffin tissue section; and the step of removing endogenous peroxidase in the tissue section.

[0054] A multi-color immunofluorescence imaging method, comprising: imaging the sample processed by the above multi-color immunofluorescence staining method.

[0055] The method for mounting a slide comprises the following steps:

[0056] a) removing the residual washing solution on the slide, adding DAPI working solution dropwise, and incubating at room temperature.

[0057] b) immersing the slide in 1x TBST buffer for 3 minutes at room temperature.

[0058] c) washing the slide with sterilized water for 2 minutes.

[0059] d) after the slide is slightly dried, adding super-strong anti-quenching mounting medium dropwise on the slide using a pipette to immerse the sample area.

[0060] e) covering the slide.

[0061] The technical scheme of the present application has the following advantages:

[0062] 1. The compound shown in formula (III) in the present application is applied in multi-color immunofluorescence staining; or, the compound shown in formula (III) is applied in the preparation of a multi-color immunofluorescence staining product; using the staining method, a plurality of different fluorescent dyes can be labeled on a plurality of antigens in a sample, and then the fluorescent dye of the previous round of staining can be eluted through an elution process, so that a new round of multi-color immunofluorescence labeling can be started. The labeled sample can be used for imaging by instruments and equipment such as a fluorescence microscope or a laser confocal microscope, and after software synthesis, the sample labeled with a plurality of different colors can be displayed. The colors are clear, bright and do not bleed. The present application not only retains the sensitivity and staining intensity of the TSA-based fluorescent staining method, but also overcomes the limitation of the number of multi-color fluorescent labels.

[0063] 2、The fluorescent dye used in the present application is covalently bound to the target to be detected, so that the sensitivity of multicolor fluorescent staining is retained, and the staining intensity is also retained.

[0064] 3、The present application provides a compound shown in formula (III). By breaking the disulfide bond (-S-S-) of the dye with a chemical reagent such as β-mercaptoethanol (β-ME), dithiothreitol (DTT) or tris (2-carboxyethyl) phosphine (TCEP), the fluorescent dye of the previous staining is removed, and the fluorescent staining can be continued, so that the target of multicolor fluorescent staining can be achieved even if only one fluorescent dye is used. For example, 10-20 targets can be detected on one slice using FITC, CY3 and CY5 fluorescent dyes.

[0065] 4、The present application provides a multicolor immunofluorescent staining method, which uses a small number of fluorescent dyes, can effectively avoid the cross-color interference between the fluorescent dyes, the staining result is accurate, and the generation of false positive results is avoided.

[0066] Using the method of the present application can avoid the influence of species pairing on staining, different protein staining can use the same species source of primary antibody and secondary antibody, the requirement of antibody species is reduced, the source of antibody is expanded, and the multicolor fluorescent staining is more easy. BRIEF DESCRIPTION OF DRAWINGS

[0067] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0068] Figure 1 is the test flow chart of example 1 of the present application;

[0069] Figure 2 is the staining result of PANCK on tonsil tissue in example 1 of the present application;

[0070] Figure 3 is the staining result of FoxP3 on tonsil tissue in example 1 of the present application;

[0071] Figure 4 is the staining result of CD31 on tonsil tissue in example 1 of the present application;

[0072] Figure 5 is the staining result of CD8a on tonsil tissue in example 1 of the present application;

[0073] Figure 6 is the staining result of Ki67 on tonsil tissue of Example 1 of the present application;

[0074] Figure 7 is the staining result of Ki67 on tonsil tissue of Example 1 of the present application; Figures 2-6 Result after software merging. DETAILED DESCRIPTION

[0075] The preparation method of 5% BSA PBS solution is to dissolve 5 grams of BSA in 100 ml of PBS solution.

[0076] 1 ml of 30% hydrogen peroxide solution is dissolved in 9 ml of PBS solution to obtain a 3% hydrogen peroxide PBS solution.

[0077] Similarly, 1 ml of 30% hydrogen peroxide solution is dissolved in 29 ml of PBS solution to obtain a 1% hydrogen peroxide PBS solution.

[0078] The basic antigen retrieval solution is a Tris-EDTA retrieval solution containing 10 mM Tris and 1 mM EDTA, with a pH of 9.

[0079] Example 1

[0080] On human tonsil paraffin sections, 2 color fluorescent dyes were used, green fluorescent dye FITC-SS-Tyr (compound of formula (I)) for 3 times of staining and red fluorescent dye CY3-SS-Tyr (compound of formula (II)) for 2 times of staining, and 5 kinds of proteins, PAN-CK, FoxP3, CD31, CD8a and Ki67, were detected on human tonsil paraffin sections in turn, and finally 5 kinds of multi-color immunofluorescence labeling results were obtained, and the test flow chart is as follows Figure 1 .

[0081]

[0082]

[0083] The 5 kinds of multi-color immunofluorescence labeling are carried out according to the general staining steps of the multi-color fluorescent staining method:

[0084] 1) Hydration after deparaffinization

[0085] a) Fresh xylene immersion sectioning (human tonsil paraffin tissue sections in this example) for 10 min, repeated 3 times.

[0086] b) Gradient ethanol immersion sectioning: 100% (v / v) ethanol for 5 min; 95% (v / v) ethanol for 5 min; 70% (v / v) ethanol for 2 min.

[0087] c) Wash the sections with sterile water for 1 min, repeat 3 times.

[0088] d) Immerse the sections in 10% (v / v) neutral formalin for 10 min.

[0089] e) Wash the sections with sterile water for 1 min, repeat 3 times.

[0090] 2) Remove endogenous peroxidase

[0091] a) Treat the sections with 1% to 3% (1% in this example) hydrogen peroxide in PBS to remove endogenous peroxidase

[0092] b) Immerse the sections in 1 x TBST buffer for 3 min, repeat 1 time.

[0093] First round, first staining:

[0094] 3) Microwave antigen retrieval

[0095] a) Place the deparaffinized hydrated sections (tissue sections) in a retrieval cup and immerse in basic antigen retrieval solution.

[0096] b) Place the retrieval cup in a microwave oven (Midea) and boil on high.

[0097] c) Maintain on low for 15 min;

[0098] d) Remove and cool to room temperature naturally.

[0099] 4) Blocking

[0100] a) Remove residual wash solution from the sections.

[0101] b) Circle the sample area on the sections with a histological pen and add blocking solution (5% BSA in PBS) to cover the sample area.

[0102] c) Maintain at room temperature with shaking for 20 min.

[0103] 5) Primary antibody incubation

[0104] a) Remove the blocking solution from the sections.

[0105] b) Add diluted primary antibody solution (PAN-CK antibody) to the sample area with a pipette.

[0106] c) Incubate at room temperature with shaking for 1 hr (optimization adjustments may be needed for different antibodies).

[0107] d) Immerse the sections in 1 x TBST buffer for 3 min, repeat 1 time.

[0108] 6) Secondary antibody incubation

[0109] a) Remove residual wash solution on the slide.

[0110] b) Directly add HRP labeled secondary antibody working solution (HRP labeled antibody matched with the species of primary antibody PAN-CK, see Table 1 for details) to the sample area.

[0111] c) Incubate at room temperature for 10 min.

[0112] d) Wash the slide with 1x TBST buffer for 3 min, repeat once.

[0113] 7) Fluorescent staining to amplify the signal (i.e. FITC-ss-Tyr color development or CY3-ss-Tyr color development)

[0114] a) Remove residual wash solution on the slide.

[0115] b) Add 100ul of dye working solution (i.e. FITC-ss-Tyr solution) with a concentration of 2ug / ml to the slide using a pipette, immerse the sample area.

[0116] c) Incubate at room temperature for 10 min.

[0117] d) Wash the slide with 1x TBST buffer for 3 min at room temperature. Repeat step d) 3 times.

[0118] e) Remove non-specific binding of antigen-antibody and dye that have been bound in steps 2) a to d.

[0119] f) Wash the slide with sterile water once, and immerse the slide in 1x TBST buffer for 2 min.

[0120] After single staining, the slide can be mounted for observation or proceed to the first round of second staining. In this example, the first round of second staining is performed.

[0121] First round of second staining: keep other parameters in steps 3) to 7) unchanged, replace the primary antibody with FoxP3 antibody, replace the secondary antibody with HRP labeled secondary antibody matched with the species of FoxP3 (see Table 1 for details), and replace the FITC-ss-Tyr solution with CY3-ss-Tyr solution.

[0122] 8) Image acquisition

[0123] Use imaging equipment such as fluorescent microscope or laser confocal microscope to collect the fluorescent signal of the sample. In this example, a fluorescent microscope is used.

[0124] 9) Remove the last round of staining with β-ME

[0125] a) soak the sections in TBST solution containing 0.1% (v / v) to 10% (v / v) of β-ME, DTT or TECP, in this example, 10% (v / v) of β-ME, to fully break the disulfide bond (-S-S-) of the dye;

[0126] b) wash the slides in 1 x TBST buffer for 3 min at room temperature. Repeat step c) for 3 times. Wash away the broken fluorescent dye. If the mounting step is included, it can also be performed before step a) as follows:

[0127] Remove the coverslips, wash the slides in 1 x TBST buffer for 3 min at room temperature, and wash away the mounting reagent.

[0128] Second round of staining: repeat steps 3) to 9) once to continue the fluorescent staining of the sections; according to Figure 1 Replace the primary antibody, secondary antibody and dye solution.

[0129] Third round of staining: repeat steps 3) to 7) once to continue the fluorescent staining of the sections; according to Figure 1 Replace the primary antibody, secondary antibody and dye solution.

[0130] Finally, read the slides, observe and interpret the stained tissue sections under the fluorescent microscope. The staining results of each protein are shown in Figures 2-6 , and Figures 2-6 The results after software stitching are shown in Figure 7 .

[0131] Table 1 Information of antibodies used in the examples

[0132]

[0133]

[0134] It is apparent that the above examples are only for the purpose of illustration, and are not intended to limit the embodiments. Based on the above description, those skilled in the art can make other different forms of changes or modifications. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or modifications derived therefrom are still within the protection scope of the present application.

Claims

1. Use of a compound represented by the following formula (III) in multicolor immunofluorescence labeling; or, use of a compound represented by the following formula (III) in preparation of a multicolor immunofluorescence staining product; Formula (III); wherein R5 is a fluorescent dye; R1, R2 in formula (III) are independently selected from any one of hydrogen, methyl, ethyl and hydroxyl; R5 is selected from any one of coumarin, cyanine dye, fluorescein, rhodamine, Bodipy and NBD amine and other code series dyes; R3 is selected from -CH2-CH2-, -C=C-, amide bond, ester bond; R4 is selected from -CH2-CH2-, -C=C-, amide bond, ester bond.

2. The use according to claim 1, wherein the coumarin dye is any one of coumarin and its derivatives; Or, the cyanine dye is any one of CY2, CY3, CY5, CY5.5, CY7 and CY7.5; Or, the fluorescein dye is any one of FAM, HEX, JOE and TET series dyes; Or, the rhodamine dye is any one of rhodamine B, rhodamine 6G and rhodamine 101 series dyes; Or, the Bodipy dye is any one of fluorinated boron dipyrromethene fluorescent dye and its derivatives; Or, the NBD amine dye is any one of benzofurazan compound and its derivatives.

3. The compound of any one of claims 1-2. Comprising the following steps: after the target protein is stained with the compound of claim 3, the disulfide bond in the compound is broken by β-mercaptoethanol, DTT or TECP, and reduced to -SH.

4. A method of multicolor immunofluorescent staining, characterized by, Comprising the following steps:

5. The method of claim 4, wherein the method is a multicolor immunofluorescent staining method. S1: first round of staining S11: first time staining of the first round; S111: A protein specific antibody incubates the sample to obtain sample 111; S112: Anti-A protein specific antibody incubates sample 111 to obtain sample 112; S113: The A protein of sample 112 is stained with the compound represented by formula (III) in claim 1; S12: second time staining of the first round S121: B protein specific antibody incubates the sample to obtain sample 121; S122: Anti-B protein specific antibody incubates sample 121 to obtain sample 122; S123: The B protein of sample 122 is stained with the compound represented by formula (III) in claim 1; S13: image acquisition; S14: the disulfide bond in the compound is broken by β-mercaptoethanol, DTT or TECP, and reduced to -SH; S2: second round of staining S21: first time staining of the second round; S211: C protein specific antibody incubates the sample to obtain sample 211; S212: Anti-C protein specific antibody incubates sample 211 to obtain sample 212; S213: The C protein of sample 212 is stained with the compound represented by formula (III) in claim 1; S22: second time staining of the second round; S221: D protein specific antibody incubates the sample to obtain sample 21; S222: Anti-D protein specific antibody incubates sample 211 to obtain sample 212; ​ S223: staining the D protein of the sample 212 with the compound of formula (III) in claim 1; S23: collecting images; S24: breaking the disulfide bond in the compound with β-mercaptoethanol, DTT or TECP, and reducing it to -SH; Similarly, the third round to the Nth round of staining is performed; N is an integer greater than or equal to 3 and less than or equal to 10; The Nth round of staining includes any one of the following: 1) the first Nth round of staining and the second Nth round of staining; 2) the first Nth round of staining; The colors of the fluorescent dyes of the compound of formula (III) used in the two staining processes in any one of the first to Nth round of staining are different.

6. The method of claim 5, wherein, After the step of "breaking the disulfide bond in the compound with β-mercaptoethanol, DTT or TECP, and reducing it to -SH" in any one of the first to Nth round of staining, a step of adding a blocking solution is further included.

7. The method according to claim 5 or 6, characterized in that, When the sample is a paraffin tissue section, the method further includes a step of deparaffinizing and hydrating the paraffin tissue section; and a step of removing endogenous peroxidase in the tissue section.

8. A method of multicolor immunofluorescence imaging, characterized by, It comprises: Imaging the sample treated by the multi-color immunofluorescence staining method according to any one of claims 4-7.

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