Dyeing method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a dyeing method and application thereof. The staining method comprises the following steps: placing a staining reagent or one or two of the staining reagent and an intermediate on a biological sample in the form of a liquid film; compared with a traditional dyeing method and a microfluidic dyeing method, the method can greatly improve the dyeing efficiency, shorten the dyeing time and save the dyeing reagent on the premise of keeping the dyeing effect. The method has wide potential application value, including fluorescent antibody dyeing, nucleotide coupled antibody dyeing, in-situ hybridization, aptamer dyeing and the like. The method can be used for animal and plant cell distribution, construction of tumor and disease maps, and screening and discovery of digital pathology and biomarkers.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a staining method and its application. Background Technology

[0002] Immunohistochemistry (IHC) / tissue immunofluorescence (IF) refers to the method of detecting, locating, and / or quantifying antigens (e.g., proteins) in biological samples using antibodies specific to a particular antigen. IHC / IF has the substantial advantage of accurately identifying specific protein sites in biological samples. It is also an effective method for examining the tissue itself. In situ hybridization (ISH) refers to the method of detecting, locating, and quantifying nucleic acids. Both IHC and ISH can be used for a variety of biological samples, such as tissues (e.g., fresh, frozen, formalin-fixed, paraffin-embedded) and cytological samples. Regardless of whether the target is nucleic acid or antigen, various markers (e.g., fluorescent, luminescent markers) can be used to detect and identify the target.

[0003] Fluorescence microscopy is a key technology in the life sciences. State-of-the-art omics approaches combine fluorescence microscopy with sophisticated algorithms to visualize tens of thousands of features within millions of pixels of a sample. These omics approaches require precise control of temperature, reagent application, and image acquisition parameters over iterative chemistry and imaging cycles lasting days or weeks. Automated execution of these methods enables robust and reproducible data generation. Traditional manual staining and microfluidic immunofluorescence techniques cannot achieve low-volume staining due to issues such as evaporation, channel size, and fluid uniformity: 1) Traditional immunofluorescence staining methods consume approximately 50–1000 μL of antibody. Furthermore, due to issues like reaction uniformity and liquid evaporation, traditional staining incubation times are long (30 minutes to 1 hour) and require a humid environment (such as a closed reaction chamber with liquid). 2) Due to the large residual volume of liquid in the flow path and the relatively thick flow channels, microfluidic immunofluorescence staining methods generally cannot achieve a volume consumption below 200 μL: a) Lunaphore (patent CN108603879A) has a fluid volume of approximately 150 μL, b) Akoya (patent CN113614244A) has a fluid volume of approximately 200 μL. Therefore, it is necessary to develop a method that can achieve low-volume staining. Summary of the Invention

[0004] The first aspect of the present invention is to provide a staining method.

[0005] A second aspect of the present invention is to provide an imaging method.

[0006] A third aspect of the present invention is to provide a biological sample staining system.

[0007] The fourth aspect of this invention is to provide an imaging system.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A first aspect of the present invention provides a staining method.

[0010] b1)~b2) Any staining method:

[0011] b1) A staining method comprising the following steps:

[0012] Place the staining reagent onto the biological sample;

[0013] The staining reagent is placed in the biological sample in the form of a liquid film;

[0014] The staining reagent is a dye-labeled substance that specifically binds to target molecules in the biological sample;

[0015] b2) A staining method comprising the following steps:

[0016] The intermediate is placed on the biological sample;

[0017] Place the staining reagent onto the biological sample;

[0018] The intermediate is placed on the biological sample in the form of a liquid film, and / or

[0019] The staining reagent is placed on the biological sample in the form of a liquid film;

[0020] The intermediate is a substance that specifically binds to the target molecule in the biological sample.

[0021] The staining reagent is a substance that specifically binds to the intermediate and is labeled with a dye.

[0022] Preferably, the thickness of the liquid film described in b1) and b2) is 5 to 60 μm.

[0023] Preferably, the liquid film described in b1) and b2) is obtained by reagent printing.

[0024] Preferably, the liquid film described in b1) and b2) is obtained by reagent spraying.

[0025] Preferably, after the intermediate described in b2) is placed on the biological sample in the form of a liquid film, a step of preventing the intermediate from evaporating is further included.

[0026] Preferably, after the staining reagent described in b1) and b2) is placed on the biological sample in the form of a liquid film, a step to prevent the staining reagent from evaporating is further included.

[0027] Preferably, the step of preventing the intermediate from evaporating is to place a cap on the liquid film.

[0028] Preferably, the thickness of the lid is 20–1200 μm.

[0029] Preferably, the step of preventing the dyeing reagent from evaporating is to place a cap on the liquid film.

[0030] Preferably, the thickness of the lid is 20–1200 μm.

[0031] Preferably, steps b1) and b2) when exchanging different reagents on the biological sample also include a cleaning step.

[0032] Preferably, the staining reagent described in b1) comprises at least one of the following: a dye-labeled first antibody or antigen-binding fragment, a dye-labeled oligonucleotide, or a dye-labeled aptamer.

[0033] Preferably, in b2):

[0034] The intermediate is a first antibody or antigen-binding fragment, and the staining reagent is a dye-labeled second antibody or antigen-binding fragment; or

[0035] The intermediate is a hapten-labeled oligonucleotide, and the staining reagent is a dye-labeled anti-hapten antibody; or

[0036] The intermediate is a first click chemical reaction module, and the staining reagent is a dye-labeled second click chemical reaction module; or

[0037] The intermediate is a first oligonucleotide-conjugated antibody, and the staining reagent is a dye-labeled second oligonucleotide; or

[0038] The intermediate is a biotin-avidin module conjugated antibody (e.g., biotin-conjugated antibody), and the staining reagent is another corresponding reaction module labeled with a dye (e.g., dye-labeled avidin).

[0039] Preferably, the biological sample comes from a single-celled organism or a multicellular organism.

[0040] Preferably, the biological sample is an organ, tissue, or cell.

[0041] Preferably, the dye comprises at least one of a luminescent substance and a fluorescent substance; further comprising at least one of fluorescein isothiocyanate, 6-carboxyfluorescein, 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein succinimide, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxytetramethylrhodamine, 6-carboxy-X-rhodamine, 5-carboxyrhodamine-6G, 6-carboxyrhodamine-6G, rhodamine 110, rhodamine B, pyrene, Cy3, Cy5, Cy7, coumarin, 7-(diethylamino)coumarin, 6-(4,6-dichlorotriazinyl)aminofluorescein, Texas Red, naphthalenefluorescein, AF532, AF488, and AF647.

[0042] Preferably, the staining method includes the following steps:

[0043] a1) Place the intermediate on a biological sample and perform the first incubation;

[0044] a2) Place the staining reagent on the biological sample and perform a second incubation;

[0045] The intermediate is placed on the biological sample in the form of a liquid film, and / or

[0046] The staining reagent is placed on the biological sample in the form of a liquid film.

[0047] Preferably, the temperature for the first incubation is 20–42°C.

[0048] Preferably, the first incubation period is 5 to 60 minutes.

[0049] Preferably, the temperature for the second incubation is 20–42°C.

[0050] Preferably, the second incubation time is 5 to 60 minutes.

[0051] Preferably, the intermediate and / or staining reagent is diluted with a diluent.

[0052] Preferably, placing the intermediate in the form of a liquid film before the biological sample further includes the following step: sealing the biological sample.

[0053] Preferably, the biological sample is permeated before the sealing process.

[0054] Preferably, the permeabilization process further includes the following steps prior to the biological sample: slicing, baking, dewaxing, hydration, and / or antigen retrieval.

[0055] Preferably, the biological sample comes from a single-celled organism or a multicellular organism.

[0056] Preferably, the biological sample is an organ, tissue, or cell.

[0057] A second aspect of the present invention provides an imaging method for imaging stained biological samples;

[0058] The stained biological sample is obtained by the staining method of the first aspect of the present invention.

[0059] Preferably, the imaging process is performed using a fluorescence microscope or a bright-field microscope.

[0060] Preferably, the imaging process further includes a pre-imaging process step before the imaging process.

[0061] Preferably, the pre-imaging process specifically involves adding an imaging buffer solution.

[0062] A third aspect of the present invention provides a biological sample staining system for performing the staining method of the first aspect of the present invention.

[0063] Preferably, the staining system comprises:

[0064] Liquid film printing or spraying module: It is used to place the intermediates and / or staining reagents onto the biological sample in the form of a liquid film.

[0065] Preferably, the staining system further comprises:

[0066] Module for preventing liquid film evaporation: It is used to prevent the liquid film from evaporating.

[0067] Preferably, the staining system further comprises:

[0068] Sealing module: It is used to seal the biological sample.

[0069] Preferably, the system further comprises:

[0070] Permeation module: It is used to permeate the biological sample.

[0071] Preferably, the system further comprises:

[0072] Rinsing module: It is used to clean the biological sample during different reagent exchanges to promote liquid exchange.

[0073] Preferably, the system further includes:

[0074] Preprocessing module: It is used to perform slicing, baking, dewaxing, hydration, and / or antigen retrieval treatment on the biological samples.

[0075] Preferably, the staining system is an automated system.

[0076] A fourth aspect of the present invention provides an imaging system for performing the imaging method of the second aspect of the present invention.

[0077] Preferably, the imaging system includes modules of the staining system of the third aspect of the present invention.

[0078] Preferably, the imaging system further comprises:

[0079] Imaging module: It is used to perform imaging processing on the stained biological sample.

[0080] Preferably, the imaging system further comprises:

[0081] Pre-imaging module: It is used to perform pre-imaging processing on the stained biological sample.

[0082] Preferably, the imaging system is an automated system.

[0083] The beneficial effects of this invention are:

[0084] This invention provides a staining method by placing a staining reagent, or a combination of a staining reagent and one or more intermediates, onto a biological sample in the form of a liquid film. Compared with traditional staining methods and microfluidic staining methods, this method can significantly improve staining efficiency, shorten staining time, and save staining reagents while maintaining staining effectiveness. This method has broad potential applications, including fluorescent antibody staining, nucleotide-coupled antibody staining, in situ hybridization, and aptamer staining. It can be used for constructing animal and plant cell distribution maps, tumor and disease atlases, as well as for digital pathology and the screening and discovery of biomarkers. Attached Figure Description

[0085] Figure 1 This is a visual representation of the fluorescent staining in Example 1 (printed staining) and Comparative Example 1 (manual staining).

[0086] Figure 2 This is a graph showing the signal-to-noise ratio of fluorescent staining in Example 1 (printed staining) and Comparative Example 1 (manual staining), where ns indicates p > 0.05.

[0087] Figure 3 These are visual diagrams of the fluorescent staining in Examples 2-5.

[0088] Figure 4 These are fluorescence intensity diagrams of the fluorescent staining in Examples 2-5. Detailed Implementation

[0089] A first aspect of the present invention provides a staining method.

[0090] b1)~b2) Any staining method:

[0091] b1) A staining method comprising the following steps:

[0092] Place the staining reagent onto the biological sample;

[0093] The staining reagent is placed in the biological sample in the form of a liquid film;

[0094] The staining reagent is a dye-labeled substance that specifically binds to target molecules in the biological sample;

[0095] b2) A staining method comprising the following steps:

[0096] The intermediate is placed on the biological sample;

[0097] Place the staining reagent onto the biological sample;

[0098] The intermediate is placed on the biological sample in the form of a liquid film, and / or

[0099] The staining reagent is placed on the biological sample in the form of a liquid film;

[0100] The intermediate is a substance that specifically binds to the target molecule in the biological sample;

[0101] The staining reagent is a substance that specifically binds to the intermediate and is labeled with a dye.

[0102] Preferably, the thickness of the liquid film in b1) and b2) is 5-60 μm; further, 7-56 μm; and even further, 20-56 μm (wherein, in b2), the thickness of the liquid film of the intermediate and the thickness of the liquid film of the staining reagent are each independently 5-60 μm; further, 7-56 μm; and even further, 20-56 μm).

[0103] Preferably, the liquid film described in b1) and b2) is obtained by reagent printing.

[0104] Preferably, the liquid film described in b1) and b2) is obtained by reagent spraying.

[0105] Preferably, the liquid film described in b1) and b2) is prepared by a liquid film printer.

[0106] Preferably, the liquid film described in b1) and b2) is prepared by a reagent nozzle.

[0107] Preferably, the liquid film printer is an Origen BP4000.

[0108] Preferably, after the intermediate described in b2) is placed on the biological sample in the form of a liquid film, a step of preventing the intermediate from evaporating is further included.

[0109] Preferably, after the staining reagent described in b1) and b2) is placed on the biological sample in the form of a liquid film, a step to prevent the staining reagent from evaporating is further included.

[0110] Preferably, the step of preventing the intermediate from evaporating is to place a cap on the liquid film.

[0111] Preferably, the thickness of the cover is 20–1200 μm; more preferably 25–1000 μm.

[0112] Preferably, the step of preventing the dyeing reagent from evaporating is to place a cap on the liquid film.

[0113] Preferably, the thickness of the cover is 20–1200 μm; more preferably 25–1000 μm.

[0114] Preferably, steps b1) and b2) when exchanging different reagents on the biological sample also include a cleaning step (e.g., step b2) includes a cleaning step before placing the staining reagent on the biological sample and after placing the intermediate on the biological sample).

[0115] Preferably, the cleaning step involves rinsing the biological sample with a cleaning solution.

[0116] Preferably, the staining reagent in b1) comprises at least one of the following: a dye-labeled first antibody or antigen-binding fragment (the antibody or antigen-binding fragment in the dye-labeled first antibody or antigen-binding fragment specifically binds to the target molecule in the biological sample), a dye-labeled oligonucleotide (the oligonucleotide in the dye-labeled oligonucleotide specifically binds to the target molecule in the biological sample (at least a portion hybridizes)), and a dye-labeled aptamer (the aptamer in the dye-labeled aptamer specifically binds to the target molecule in the biological sample). (i.e., the substance specifically binding to the target molecule in the biological sample in b1) comprises at least one of the following: a first antibody or antigen-binding fragment, an oligonucleotide, and an aptamer. Further, the substance specifically binding to the target molecule in the biological sample in b1 (i.e., the dye-labeled first antibody, the dye-labeled oligonucleotide, or the dye-labeled aptamer) is a first antibody, an oligonucleotide, or an aptamer.

[0117] Preferably, in b2):

[0118] The intermediate is a first antibody or antigen-binding fragment, and the staining reagent is a dye-labeled second antibody or antigen-binding fragment; or

[0119] The intermediate is an oligonucleotide labeled with a hapten (e.g., biotin, digoxigenin), and the staining reagent is a dye-labeled anti-hapten antibody; or

[0120] The intermediate is a first click chemical reaction module, and the staining reagent is a dye-labeled second click chemical reaction module; or

[0121] The intermediate is a first oligonucleotide-conjugated antibody, and the staining reagent is a dye-labeled second oligonucleotide; or

[0122] The intermediate is a biotin-avidin (preferably streptavidin) module conjugated antibody, and the staining reagent is a dye-labeled corresponding reaction module (e.g., the intermediate is a biotin-conjugated antibody and the staining reagent is dye-labeled avidin (preferably streptavidin); the intermediate is an avidin (preferably streptavidin) conjugated antibody and the staining reagent is dye-labeled biotin).

[0123] Preferably, the intermediate in b2) is a first antibody or antigen-binding fragment, and the staining reagent is a dye-labeled second antibody or antigen-binding fragment.

[0124] Preferably, when the staining reagent in b1) contains an oligonucleotide, the oligonucleotide hybridizes with at least a portion of the target molecule in the biological sample that specifically binds to it; more preferably, the oligonucleotide is at least 70%, 80%, 90%, or 95% complementary to the nucleotide sequence of the target molecule in the biological sample that specifically binds to it.

[0125] Preferably, when the intermediate in b2) comprises an oligonucleotide, the oligonucleotide of the intermediate hybridizes with at least a portion of the oligonucleotide that specifically binds to the target molecule in the biological sample or the staining reagent; more preferably, the nucleotide sequences of the oligonucleotide of the intermediate and the oligonucleotide that specifically binds to the target molecule in the biological sample or the staining reagent are at least 70%, 80%, 90%, or 95% complementary.

[0126] Preferably, the biological sample is any solid or fluid biological sample obtained from, excreted or secreted by, any living organism, including but not limited to, single-celled organisms (such as bacteria, yeast, protozoa, and amoebas) and multicellular organisms (such as plants or animals), including samples from healthy or apparently healthy human subjects or human patients affected by a symptom or disease to be diagnosed or studied, such as cancer. For example, the biological sample may be a biological fluid or any bodily secretion, transudate, exudate (e.g., fluid obtained from an abscess or any other site of infection or inflammation) obtained from, for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, aqueous humor, or vitreous humor, or fluid obtained from a joint (e.g., a normal joint or a joint affected by disease). The biological sample may also be a sample obtained from any organ or tissue (including biopsy or autopsy samples, such as tumor biopsies), or may include cells (whether primary or cultured cells) or a culture medium regulated by any cell, tissue, or organ.

[0127] Preferably, the biological sample can be fresh, frozen, or fixed.

[0128] Preferably, the biological sample may include formalin-fixed paraffin-embedded (FFPE) tissue sections, frozen tissue sections, fresh tissue, cells obtained from the subject (e.g., via fine needle aspiration or other techniques), cultured cells, biological tissue, biological fluid, homogenate, or unknown biological sample; further, formalin-fixed paraffin-embedded (FFPE) tissue sections or formalin-fixed paraffin-embedded (FFPE) cell sections.

[0129] Preferably, the target molecule comprises an antigen, a nucleotide, or other molecular marker.

[0130] Preferably, the dye comprises at least one of a luminescent substance and a fluorescent substance; further comprising fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (commonly abbreviated as FAM and F), 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein succinimide ester (6-HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE or J), 6-carboxytetramethylrhodamine (6-TAMRA), 6-carboxy-X - At least one of the following: Rhodamine (6-ROX), 5-carboxyrhodamine-6G (5-CR6G), 6-carboxyrhodamine-6G (6-CR6G), Rhodamine 110, Rhodamine B, Pyrene, Cy3, Cy5, Cy7, Coumarin, 7-(diethylamino)coumarin, 6-(4,6-dichlorotriazinyl)aminofluorescein (6-TET), Texas Red, Naphthalenefluorescein, AF532, AF488, AF647; further, AF532.

[0131] Preferably, the staining method includes the following steps:

[0132] a1) Place the intermediate on a biological sample and perform the first incubation;

[0133] a2) Place the staining reagent on the biological sample and perform a second incubation;

[0134] The intermediate is placed on the biological sample in the form of a liquid film, and / or

[0135] The staining reagent is placed on the biological sample in the form of a liquid film.

[0136] Preferably, the staining method includes the following steps:

[0137] a1) The intermediate is placed on the biological sample in the form of a liquid film for the first incubation;

[0138] a2) Place the staining reagent onto the biological sample in the form of a liquid film and perform a second incubation.

[0139] Preferably, the thickness of the liquid film described in a1) and a2) is independently selected from 5 to 60 μm; further, it is 7 to 56 μm; and even further, it is 20 to 56 μm.

[0140] Preferably, the liquid film described in a1) and a2) is obtained by reagent printing.

[0141] Preferably, the liquid film described in a1) and a2) is obtained by reagent spraying.

[0142] Preferably, the liquid film described in a1) and a2) is prepared by a liquid film printer.

[0143] Preferably, the liquid film described in a1) and a2) is prepared by a reagent nozzle.

[0144] Preferably, the liquid film printer is an Origen BP4000.

[0145] Preferably, after the intermediate is placed on the biological sample in the form of a liquid film, the step further includes preventing the intermediate from evaporating.

[0146] Preferably, after the staining reagent is placed on the biological sample in the form of a liquid film, the process further includes a step to prevent the staining reagent from evaporating.

[0147] Preferably, the step of preventing the intermediate from evaporating is to place a cap on the liquid film.

[0148] Preferably, the thickness of the cover is 20–1200 μm; more preferably 25–1000 μm.

[0149] Preferably, the step of preventing the dyeing reagent from evaporating is to place a cap on the liquid film.

[0150] Preferably, the thickness of the cover is 20–1200 μm; more preferably 25–1000 μm.

[0151] Preferably, the temperature for the first incubation is 20–42°C; further, it is 25–42°C; and even further, it is 35–39°C.

[0152] Preferably, the first incubation time is 5-60 min; further, 8-30 min; even further, 8-12 min; and still further, 10 min.

[0153] Preferably, the temperature of the second incubation is 20–42°C; further, it is 25–42°C; and even further, it is 35–39°C.

[0154] Preferably, the second incubation time is 5-60 min; further, 8-30 min; even further, 8-12 min; and still further, 10 min.

[0155] Preferably, the intermediate (preferably a first antibody or antigen-binding fragment) and / or staining reagent (a dye-labeled second antibody or antigen-binding fragment) are diluted with a diluent.

[0156] Preferably, the diluent is a buffer solution.

[0157] Preferably, placing the intermediate in the form of a liquid film before the biological sample further includes the following step: sealing the biological sample.

[0158] Preferably, the sealing process specifically involves adding a sealing agent (i.e., mixing a sealing agent (such as BSA, gelatin, serum, skim milk, etc.) with the biological sample).

[0159] Preferably, the biological sample is permeated before the sealing process.

[0160] Preferably, the permeation treatment specifically involves adding a permeation solution (such as Triton X-100) (i.e., mixing the permeation solution with the biological sample).

[0161] Preferably, the permeabilization process further includes the following steps before the biological sample: slicing, baking, dewaxing, hydration, and / or antigen retrieval (these steps are conventional methods in the art).

[0162] Preferably, the process of exchanging different reagents on the biological sample further includes a cleaning step (e.g., a1) and a2); and the process of permeabilizing the biological sample and placing the intermediate in the form of a liquid film before the biological sample further includes a cleaning step.

[0163] Preferably, the cleaning step involves rinsing the biological sample with a cleaning solution.

[0164] A second aspect of the present invention provides an imaging method for imaging stained biological samples;

[0165] The stained biological sample is obtained by the staining method of the first aspect of the present invention.

[0166] Preferably, the imaging process is performed using a fluorescence microscope or a bright-field microscope.

[0167] Preferably, the imaging process further includes a pre-imaging process step before the imaging process.

[0168] Preferably, the pre-imaging process specifically involves adding an imaging buffer (i.e., mixing the imaging buffer with the stained biological sample).

[0169] Preferably, the biological sample is further subjected to a cleaning step when different reagents are exchanged (e.g., after the staining reagent is placed on the biological sample (preferably after the second incubation) and before the pre-imaging process).

[0170] Preferably, the cleaning step involves rinsing the biological sample with a cleaning solution.

[0171] A third aspect of the present invention provides a biological sample staining system for performing the staining method of the first aspect of the present invention.

[0172] Preferably, the staining system comprises:

[0173] Liquid film printing or spraying module: It is used to place the intermediates and / or staining reagents onto the biological sample in the form of a liquid film.

[0174] Preferably, the staining system further comprises:

[0175] Module for preventing liquid film evaporation: It is used to prevent the liquid film from evaporating (specifically, by placing a lid on the liquid film).

[0176] Preferably, the staining system further comprises:

[0177] Sealing module: It is used to seal the biological sample (specifically, by adding a sealing agent, i.e., mixing the sealing agent with the biological sample).

[0178] Preferably, the system further comprises:

[0179] Permeation module: It is used to permeate the biological sample (specifically, to add permeation solution, that is, to mix the permeation solution with the biological sample).

[0180] Preferably, the system further comprises:

[0181] Rinsing module: It is used to clean the biological sample during different reagent exchanges to promote liquid exchange (specifically, by adding cleaning solution to rinse the biological sample).

[0182] Preferably, the system further includes:

[0183] Preprocessing module: It is used to perform slicing, baking, dewaxing, hydration, and / or antigen retrieval treatment on the biological samples.

[0184] Preferably, the staining system is an automated system.

[0185] A fourth aspect of the present invention provides an imaging system for performing the imaging method of the second aspect of the present invention.

[0186] Preferably, the imaging system includes modules of the staining system of the third aspect of the present invention.

[0187] Preferably, the imaging system further comprises:

[0188] Imaging module: It is used to perform imaging processing on the stained biological sample (specifically, to image the stained biological sample using a fluorescence microscope or a bright-field microscope).

[0189] Preferably, the imaging system further comprises:

[0190] Pre-imaging module: It is used to pre-image the stained biological sample (specifically, by adding imaging buffer, i.e., mixing the imaging buffer with the stained biological sample).

[0191] Preferably, the imaging system is an automated system.

[0192] The present invention will be further described in detail below through specific embodiments.

[0193] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0194] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0195] Example 1: A method for imaging paraffin sections of mouse brain

[0196] A method for imaging paraffin sections of mouse brain includes the following steps:

[0197] i. The paraffin blocks of mouse brain treated with FFPE (purchased from the Experimental Animal Center of Guangdong Medical University) were sectioned using a paraffin microtome. The sections were placed in a water bath at 40-45℃ until they were fully expanded.

[0198] ii. The slides treated with surface treatment (Poly-l-lysine (Sigma#P8920), APTS (Sigma#440140), Gelatin (Sigma#V900863), Gelatin Chrome Alum, etc.; in this embodiment, Gelatin Chrome Alum) are retrieved and left to stand vertically overnight.

[0199] iii. Dewaxing is performed using xylene for 3 hours;

[0200] iv. Add anhydrous ethanol, 95% ethanol, 70% ethanol, 50% ethanol, and ultrapure water in sequence (treat each solution for 20 minutes each) for hydration treatment;

[0201] v. The slide is placed in antigen retrieval solution (Thermofisher #00-4956-58) and subjected to high temperature (95℃) for 20 minutes for antigen retrieval;

[0202] vi. Add 0.5% Triton X-100 and punch for 15 minutes;

[0203] vii. Use a pipette to draw up the cleaning solution and rinse three times;

[0204] viii. The primary antibody Rabbit anti-Aqua4 (Thermofisher, #PA5-53234) was diluted with buffer and then printed at DPI 300x300 on a 20x25mm area using a liquid film printer (Aorui BP4000, printhead frog50). The liquid film thickness was 20μm, and the total reagent consumption was approximately 10μL.

[0205] ix. Cover with a 1000μm cap with a rubber ring to prevent liquid evaporation;

[0206] Incubate at 37℃ for 10 minutes;

[0207] xi. Perform three cleaning cycles using cleaning solution;

[0208] xii. After diluting the Goat anti-Rabbit AF532 (Thermofisher, #A-11009) secondary antibody with buffer, a 20x25mm area was printed using a liquid film printer at DPI 300x300, with a liquid film thickness of 20μm. The total reagent consumption was approximately 10μL.

[0209] xiii. Repeat steps ix.-xi.;

[0210] xiv. Add imaging buffer (Tolox (Sigma #648471) 800mM) and coverslip, and image using a fluorescence microscope.

[0211] Example 2: A method for imaging paraffin sections of Jurkat cells

[0212] A method for imaging paraffin sections of Jurkat cells includes the following steps:

[0213] i. Use a paraffin microtome to section the FFPE-treated paraffin blocks of Jurkat cells (provided by Wuhan Saiwei), and place the sections in a water bath at 40-45℃ until the sections are fully developed;

[0214] ii. The slides treated with surface treatment (Poly-l-lysine (Sigma#P8920), APTS (Sigma#440140), Gelatin (Sigma#V900863), Gelatin Chrome Alum, etc.; in this embodiment, Gelatin Chrome Alum) are retrieved and left to stand vertically overnight.

[0215] iii. Dewaxing is performed using xylene for 3 hours;

[0216] iv. Add anhydrous ethanol, 95% ethanol, 70% ethanol, 50% ethanol, and ultrapure water in sequence (treat each solution for 20 minutes each) for hydration treatment;

[0217] v. The slide is placed in antigen retrieval solution (Thermofisher #00-4956-58) and subjected to high temperature (95℃) for 20 minutes for antigen retrieval;

[0218] vi. Add 0.5% Triton X-100 and punch for 15 minutes;

[0219] vii. Use a pipette to draw up the cleaning solution (same as in Example 1) and rinse 3 times;

[0220] viii. The Rabbit anti-CD3 (Sino-Chang Shenzhou, #CT026-R301) primary antibody was diluted with buffer (same as in Example 1) and printed in DPI 300x300 on a 20x25mm area using a liquid film printer. The liquid film thickness was 7μm, and the total reagent consumption was approximately 3.5μL.

[0221] ix. Cover with a 1000μm cap with a rubber ring to prevent liquid evaporation;

[0222] Incubate at 37℃ for 10 minutes;

[0223] xi. Perform cleaning three times using the cleaning solution (same as in Example 1);

[0224] xii. After diluting the Goat anti-Rabbit AF532 (Thermofisher, #A-11009) secondary antibody with buffer (as in Example 1), a 20x25mm area was printed with a DPI of 300x300 using a liquid film printer. The liquid film thickness was 7μm, and the total reagent consumption was approximately 3.5μL.

[0225] xiii. Repeat steps ix.-xi.;

[0226] xiv. Add imaging buffer (Tolox (Sigma #648471) 800mM) and coverslip, and image using a fluorescence microscope.

[0227] Example 3: A method for imaging paraffin sections of Jurkat cells

[0228] A method for imaging paraffin sections of Jurkat cells is the same as that in Example 2, except that the liquid film thickness in steps viii and xii is 14 μm and the total reagent consumption is approximately 7 μL.

[0229] Example 4: A method for imaging paraffin sections of Jurkat cells

[0230] A method for imaging paraffin sections of Jurkat cells is the same as that in Example 2, except that the liquid film thickness in steps viii and xii is 28 μm and the total reagent consumption is approximately 14 μL.

[0231] Example 5: A method for imaging paraffin sections of Jurkat cells

[0232] A method for imaging paraffin sections of Jurkat cells is the same as that in Example 2, except that the liquid film thickness in steps viii and xii is 56 μm and the total reagent consumption is approximately 28 μL.

[0233] Comparative Example 1: A method for imaging paraffin sections of mouse brain

[0234] A method for imaging paraffin sections of mouse brain includes the following steps:

[0235] 1. Elute the stained sections using antibody elution buffer:

[0236] The following examples illustrate three different schemes for multiplex fluorescent staining:

[0237] 1) Secondary antibody amplification:

[0238] i. Perform flow-through washing with PBS (time: 100 seconds, flow rate: 1200 μL / min);

[0239] ii. Add the primary antibody diluted with 1% BSA solution (1:10-1:400) and incubate at 37°C for 15-45 minutes;

[0240] iii. Perform flow-through washing with PBS (time: 100 seconds, flow rate: 1200 μL / min);

[0241] iv. Add fluorescently labeled secondary antibody diluted with 1% BSA solution (1:10-1:400) and incubate at 37 degrees Celsius for 10-30 minutes;

[0242] v. Perform flow-through washing with PBS (time: 100 seconds, flow rate: 1200 μL / min);

[0243] vi. Add imaging buffer (antioxidant solutions such as Trolox, N-acetyl cysteine, and DTT);

[0244] vii. Perform a full-slice scan at a specific wavelength using the fluorescently labeled secondary antibody;

[0245] viii. Antibody elution was performed using antibody elution buffer (weakly acidic 2ME + SDS + TrisHCl solution, acidic glycine + SDS solution, or acidic glycine + urea + GC + TCEP solution, etc.) at a temperature controlled (25-56 degrees Celsius).

[0246] ix. Perform flow-through washing with PBS (time: 100 seconds, flow rate: 1200 μL / min);

[0247] x. Perform a second blocking treatment using a blocking agent (such as 10% BSA, 5% Goat Serum, or 10% skim milk) for 30 minutes;

[0248] xi. Loop through step i to x;

[0249] 2) Fluorescently labeled primary antibody:

[0250] i. Perform flow-through washing with PBS (time: 100 seconds, flow rate: 1200 μL / min);

[0251] ii. Add fluorescently labeled primary antibody diluted with 1% BSA solution (1:10-1:400) and incubate at 37 degrees Celsius for 15-45 minutes;

[0252] iii. Perform flow-through washing with PBS (time: 100 seconds, flow rate: 1200 μL / min);

[0253] iv. Add imaging buffer (antioxidant solutions such as Trolox, N-acetyl cysteine, and DTT);

[0254] v. Perform a full-slice scan of the fluorescently labeled primary antibody at a specific wavelength;

[0255] vi. Elute antibodies for 30 minutes at a controlled temperature (25-56 degrees Celsius) using antibody elution buffer (weakly acidic 2ME + SDS + TrisHCl solution, acidic glycine + SDS solution, or acidic glycine + urea + GC + TCEP solution, etc.). Alternatively, quench fluorescence using a quencher (0.1M sodium bicarbonate + 3% hydrogen peroxide, sodium borohydride, 24mM sodium hydroxide + 4.5% hydrogen peroxide, etc.).

[0256] vii. Perform flow-through washing with PBS (time: 100 seconds, flow rate: 1200 μL / min);

[0257] viii. Perform a second blocking treatment (such as 10% BSA, 5% Goat Serum, or 10% skim milk) for 30 minutes;

[0258] ix. Loop i. to viii.

[0259] 3) Nucleotide-coupled fluorescence:

[0260] i. Perform flow-through washing with PBS (time: 100 seconds, flow rate: 1200 μL / min);

[0261] ii. Add nucleotide-conjugated primary antibody diluted with 1% BSA solution (1:10-1:400) and incubate at 37°C for 15-45 minutes;

[0262] iii. Add PFA or other fixatives for amines to fix the antibody-sample;

[0263] iv. Perform flow-through washing with PBS (time: 100 seconds, flow rate: 1200 μL / min);

[0264] v. Nucleotides coupled to the primary antibody can be amplified using methods such as RCA and PER (primer replacement method);

[0265] vi. Perform flow-through washing with PBS (time: 100 seconds, flow rate: 1200 μL / min);

[0266] vii. Add the corresponding fluorescently labeled nucleotide double strand to the solution (PBS, TBS, etc.), add the sample, and incubate for 30 minutes. Gradual cooling incubation can be performed to enhance specificity.

[0267] viii. Perform flow-through washing with PBS (time: 100 seconds, flow rate: 1200 μL / min);

[0268] ix. Add imaging buffer (antioxidant solution such as Trolox, N-acetyl cysteine, DTT, etc.);

[0269] x. Perform a full-slice scan of the fluorescence at a specific wavelength;

[0270] xi. Double-stranded DNA can be eluted by high-temperature unwinding and elution, degradation by double-stranded DNA degrading enzymes, and unwinding and elution by DNA helicases.

[0271] xii. Cycle vi. to xi; In multiple fluorescence staining protocols, the addition of a reducing agent to the reagents can help the antibodies overcome the cross-linking caused by photography, making them easier to elute; In this comparative example, antibody elution buffer (weakly acidic 2ME + SDS + TrisHCl solution) was used to elute the stained sections obtained in Example 1.

[0272] 2. Perform three washes using the same cleaning solution as in Example 1 for liquid film staining, each lasting 5 minutes;

[0273] 3. Add 200 μL of Rabbit anti-Aqua4 primary antibody diluted to the same concentration as in the liquid membrane staining in Example 1;

[0274] 4. Incubate at room temperature for 10 minutes;

[0275] 5. Perform three washes using the same washing solution as in Example 1 for liquid film staining, each lasting 5 minutes;

[0276] 6. Add 200 μL of Goat anti-Rabbit AF532 secondary antibody diluted to the same concentration as in the liquid membrane staining in Example 1;

[0277] 7. Incubate at room temperature for 10 minutes;

[0278] 8. Perform three washes using the same washing solution as in the liquid film staining in Example 1, each lasting 5 minutes;

[0279] 9. Imaging was performed using the same imaging buffer as that used for liquid membrane staining in Example 1.

[0280] Effect Implementation Examples: Verification of the Effects of the Imaging Methods (Examples)

[0281] 1) Visual diagrams of fluorescent staining in Example 1 (printed staining) and Comparative Example 1 (manual staining) are shown below. Figure 1 As shown: The results of Example 1 and Comparative Example 1 are basically similar, with slightly lower fluorescence intensity. Straight lines were drawn at the signal location and on both sides to select areas, and the fluorescence intensity at the signal location was recorded. The background signal intensity of the tissue in the adjacent area was recorded, and the difference was used to calculate the SNR. The signal-to-noise ratio of fluorescence staining in Example 1 (printed staining) and Comparative Example 1 (manual staining) is shown in the figure. Figure 2 (Multiple sampling, N=15) As shown: The signal-to-noise ratio of Example 1 and Comparative Example 1 is similar; it can be seen that this application still has a good staining effect while reducing the amount of antibody and the incubation time.

[0282] 2) Visual diagrams of fluorescent staining in Examples 2-5 are shown below. Figure 3 As shown, ImageJ was used to perform mean statistical analysis on the fluorescence intensity of cells across the entire image. The fluorescence intensity is as follows: Figure 4 As shown: the fluorescence intensity increases with increasing liquid film thickness, and the liquid film thickness (liquid film volume) is positively correlated with the fluorescence intensity (R = 0.97). Figure 4 Blank (where the antibody is replaced with an equal volume of antibody-free diluent, and the reading intensity is autofluorescence).

[0283] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. b1)~b2) Any staining method: b1) A staining method comprising the following steps: Place the staining reagent onto the biological sample; The staining reagent is placed on the biological sample in the form of a liquid film; The staining reagent is a dye-labeled substance that specifically binds to target molecules in the biological sample; b2) A staining method comprising the following steps: The intermediate is placed on the biological sample; Place the staining reagent onto the biological sample; The intermediate is placed on the biological sample in the form of a liquid film, and / or The staining reagent is placed on the biological sample in the form of a liquid film; The intermediate is a substance that specifically binds to the target molecule in the biological sample; The staining reagent is a substance that specifically binds to the intermediate and is labeled with a dye.

2. The staining method according to claim 1, characterized in that: The thickness of the liquid film described in b1) and b2) is 5–60 μm; Preferably, the liquid films described in b1) and b2) are obtained by reagent printing. Preferably, the liquid film described in b1) and b2) is obtained by reagent spraying.

3. The staining method according to claim 1 or 2, characterized in that: b2) After the intermediate is placed on the biological sample in the form of a liquid film, the step of preventing the intermediate from evaporating is further included; and / or After the staining reagent described in b1) and b2) is placed on the biological sample in the form of a liquid film, the step of preventing the staining reagent from evaporating is also included. Preferably, the step of preventing the intermediate from evaporating is: placing a cap on the liquid film; Preferably, the thickness of the lid is 20–1200 μm; Preferably, the step of preventing the dyeing reagent from evaporating is: placing a cap on the liquid film; Preferably, the thickness of the lid is 20–1200 μm; Preferably, steps b1) and b2) when exchanging different reagents on the biological sample also include a cleaning step.

4. The staining method according to claim 3, characterized in that: The staining reagent described in b1) comprises at least one of the following: a dye-labeled first antibody or antigen-binding fragment, a dye-labeled oligonucleotide, or a dye-labeled aptamer; Preferably, in b2): The intermediate is a first antibody or antigen-binding fragment, and the staining reagent is a dye-labeled second antibody or antigen-binding fragment; or The intermediate is a hapten-labeled oligonucleotide, and the staining reagent is a dye-labeled anti-hapten antibody; or The intermediate is a first click chemical reaction module, and the staining reagent is a dye-labeled second click chemical reaction module; or The intermediate is a first oligonucleotide-conjugated antibody, and the staining reagent is a dye-labeled second oligonucleotide; or The intermediate is a biotin-avidin module conjugated antibody, and the staining reagent is another corresponding reaction module labeled with a dye. Preferably, the dye comprises at least one of a luminescent substance and a fluorescent substance; further comprising at least one of fluorescein isothiocyanate, 6-carboxyfluorescein, 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein succinimide, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxytetramethylrhodamine, 6-carboxy-X-rhodamine, 5-carboxyrhodamine-6G, 6-carboxyrhodamine-6G, rhodamine 110, rhodamine B, pyrene, Cy3, Cy5, Cy7, coumarin, 7-(diethylamino)coumarin, 6-(4,6-dichlorotriazinyl)aminofluorescein, Texas Red, naphthalenefluorescein, AF532, AF488, and AF647.

5. The staining method according to claim 4, characterized in that: The staining method includes the following steps: a1) Place the intermediate on a biological sample and perform the first incubation; a2) Place the staining reagent on the biological sample and perform a second incubation; The intermediate is placed on the biological sample in the form of a liquid film, and / or The staining reagent is placed on the biological sample in the form of a liquid film; Preferably, the temperature for the first incubation is 20–42°C; Preferably, the first incubation period is 5 to 60 minutes; Preferably, the temperature for the second incubation is 20–42°C; Preferably, the second incubation time is 5 to 60 minutes; Preferably, the intermediate and / or staining reagent is diluted with a diluent; Preferably, placing the intermediate in the form of a liquid film before the biological sample further includes the following step: sealing the biological sample; Preferably, the biological sample is further permeated before the sealing process. Preferably, the permeabilization process further includes the following steps before the biological sample: slicing, baking, dewaxing, hydration, and / or antigen retrieval. Preferably, the biological sample is derived from a single-celled organism or a multicellular organism; Preferably, the biological sample is an organ, tissue, or cell.

6. An imaging method for imaging stained biological samples; The stained biological sample is obtained by the staining method according to any one of claims 1 to 5.

7. The imaging method according to claim 6, characterized in that: The imaging processing is performed using a fluorescence microscope or a bright-field microscope. Preferably, the imaging process further includes a pre-imaging process step before the imaging process; Preferably, the pre-imaging process specifically involves adding an imaging buffer solution.

8. A biological sample staining system for performing the staining method according to any one of claims 1 to 5; Preferably, the staining system comprises: Liquid film printing or spraying module: It is used to place the intermediate and / or staining reagent onto the biological sample in the form of a liquid film; Preferably, the staining system further comprises: Module for preventing liquid film evaporation: It is used to prevent the liquid film from evaporating; Preferably, the system further comprises: Permeation module: It is used to permeate the biological sample; Preferably, the staining system further comprises: Sealing module: It is used to seal the biological sample; Preferably, the system further comprises: Rinsing module: It is used to clean the biological sample during different reagent exchanges to promote liquid exchange; Preferably, the system further includes: Preprocessing module: It is used to perform slicing, baking, dewaxing, hydration, and / or antigen retrieval treatment on the biological samples; Preferably, the staining system is an automated system.

9. An imaging system for performing the imaging method of claim 6 or 7.

10. The imaging system according to claim 9, characterized in that: The imaging system includes the modules of the staining system of claim 8; Preferably, the imaging system further comprises: Imaging module: It is used to perform imaging processing on the stained biological sample; Preferably, the imaging system further comprises: Pre-imaging module: It is used to perform pre-imaging processing on the stained biological sample; Preferably, the imaging system is an automated system.

Images