CN111830256A - Immunofluorescence multiple staining kit for paraffin section and using method - Google Patents
Immunofluorescence multiple staining kit for paraffin section and using method Download PDFInfo
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Abstract
The invention provides a paraffin section immunofluorescence multiple staining kit, which comprises a reagent A, a reagent B, a diluent, an FITC reagent, a Cy3 reagent and a Cy5 reagent, and also provides a method for using the paraffin section immunofluorescence multiple staining kit, the kit adopts a TSA signal amplification technology, the principle is similar to that of common immunohistochemistry, a primary antibody is combined with an antigen, an HRP-labeled secondary antibody is incubated, the HRP is added into a fluorescein substrate of a system in a catalytic manner to generate an activated fluorescein substrate, the activated fluorescein substrate can be covalently combined with tyrosine on the antigen, so that fluorescein is stably covalently combined on a sample, then the non-covalently combined antibody is washed off by thermal restoration, a secondary primary antibody is replaced for secondary incubation, another fluorescein substrate is replaced, so that multiple labeling can be realized by reciprocating, only one antibody is incubated in each round, the non-covalently combined antibody is washed off after microwave treatment, there is no limitation on the source of the antibody species used.
Description
Technical Field
The invention relates to the technical field of biomedical application, in particular to a paraffin section immunofluorescence multiple staining kit and a using method thereof.
Background
The immunofluorescence technique is an experimental technique based on the combination of antibody and antigen specificity, and is widely applied to basic research and clinical experiments due to the advantages of simple operation, high efficiency, strong specificity and the like; with the rise of immunotherapy, more and more markers in tumor microenvironment are found to be closely related to disease treatment and progression, and the simultaneous staining of the markers and the study on the co-localization, expression amount and distance relationship among the markers become common requirements of tumor immune research; the traditional immunofluorescence labeling can realize simultaneous labeling of two antibodies, and the two antibodies are required to be prepared from different species and used for connecting two secondary antibodies of different fluorescein for color development; the experimental method has high requirements on one antigen source species, and if more than two kinds of antigen markers are needed, all the antibody source species are required to be different, so that all the antibodies meeting the requirements cannot be purchased, and multiple antigen markers cannot be carried out.
Disclosure of Invention
The invention aims to provide a paraffin section immunofluorescence multiple staining kit and a using method thereof, wherein the paraffin section immunofluorescence multiple staining kit and the using method can simultaneously mark a plurality of different antigens on the same section, and do not limit the source species of an antigen of the used antigens.
The invention provides a paraffin section immunofluorescence multiple staining kit, which comprises a reagent A, a reagent B, diluent, a FITC reagent, a Cy3 reagent and a Cy5 reagent, wherein the reagent A is a reagent A;
the reagent A is mainly Biotin-Tyramide;
the reagent B is 30% of H2O2；
The diluent is a phosphate buffer solution with the pH value of 7.4;
the FITC reagent is Streptavidin-FITC;
the Cy3 reagent is Streptavidin-Cy 3;
the Cy5 reagent is Streptavidin-Cy 5.
A method for using a paraffin section immunofluorescence multiple staining kit comprises the following steps:
s1: reagent preparation, comprising the steps of:
s1-1: dissolving 1ul of reagent A in 500ul of diluent to obtain a reagent 1;
s1-2: dissolving 1ul of reagent B in 10ul of diluent to obtain a reagent 2;
s1-3: dissolving 1ul of the reagent 2 in 100ul of the reagent 1 to prepare a multiple IF staining working solution;
s1-4: dissolving 1ul of FITC reagent in 1ml of diluent to prepare FITC working solution;
s1-5: dissolving 1ul of Cy3 reagent in 1ml of diluent to prepare Cy3 working solution;
s1-6: dissolving 1ul of Cy5 reagent in 1ml of diluent to prepare Cy5 working solution;
s2: paraffin section dewaxing to water: sequentially placing the slices in xylene I15 min, xylene II 15min, anhydrous ethanol I5 min, anhydrous ethanol II 5min, 85% ethanol 5min and 75% ethanol 5min, and washing with distilled water;
s3: antigen retrieval: placing the tissue slices in a repairing box filled with EDTA antigen repairing buffer solution, performing antigen repairing in a steamer, timing for 30min after the temperature reaches 95 ℃, placing the slides in PBS after natural cooling, and shaking and washing the slides on a decoloring shaking table for 3 times, 5min each time;
s4: circle drawing autofluorescence quenching: after the section is slightly dried, a circle is drawn around the tissue by a tissue pen to prevent the antibody from flowing away, an autofluorescence quencher A is added into the circle, the circle is incubated for 15min at room temperature, then washed by flowing water for 10min, an autofluorescence quencher B is added into the circle, the circle is incubated for 10min at room temperature, and then washed by flowing water for 10 min;
s5: sealing serum, namely dropwise adding 5% BSA in a circle, and incubating for 30min at room temperature;
s6: adding a first primary antibody: removing the confining liquid, dripping the first primary antibody into the ring, and incubating overnight at 4 ℃ in a wet box;
s7: adding a corresponding secondary HRP-labeled antibody: placing the slide in PBS, shaking and washing for 3 times on a decoloring shaking table for 5min each time, dripping a second antibody labeled by corresponding HRP in a ring to cover the tissue after the slide is slightly dried, and incubating for 1h in a room temperature wet box;
s8: kit reagents were stained using multiple IF: placing the slide in PBS, and washing for 5min each time for 3 times; dripping the prepared multiple IF staining working solution into the ring, and incubating the inner chamber of the wet box for 30min in a warm and dark place; placing the slide in PBS, and washing for 5min each time for 2 times in a manner of shaking on a decoloring shaking table in a dark place;
s9: adding FITC working solution: after the section is slightly dried, FITC working solution is dripped into the ring to cover the tissue, and the tissue is incubated for 30min in a room temperature wet box in a dark place;
s10: microwave treatment to remove excess primary antibody: placing the tissue slices in a repairing box filled with EDTA antigen repairing buffer solution in a microwave oven, stopping heating for 5min with medium fire for 5min, turning to medium fire for 10min with medium fire, preventing excessive evaporation of the buffer solution, cutting into dry slices, naturally cooling, placing the slices in PBS, and shaking and washing for 3 times with 5min each time;
s11: adding a second primary antibody: dropwise adding the diluted second primary antibody on the slices, and flatly placing the slices in a wet box for incubation overnight at 4 ℃ in a dark place;
s12: adding a corresponding secondary HRP-labeled antibody: placing the slide in PBS, shaking on a decoloring shaking table to wash in the dark for 3 times, each time for 5min, dripping a second antibody covering tissue corresponding to the HRP mark into a ring after the slide is slightly dried, and incubating in the dark for 1h in a room temperature wet box;
s13: kit reagents were stained using multiple IF: placing the slide in PBS, and washing for 5min each time for 3 times; dripping the prepared multiple IF staining working solution into the ring, and incubating the inner chamber of the wet box for 30min in a warm and dark place; placing the slide in PBS, and washing for 5min each time for 2 times in a manner of shaking on a decoloring shaking table in a dark place;
s14: adding Cy3 working solution: after the slices are slightly dried, Cy3 working solution is dripped into the ring to cover the tissues, and the tissues are incubated in a room-temperature wet box for 30min in a dark place;
s15: microwave treatment to remove excess primary antibody: placing the tissue slices in a repairing box filled with EDTA antigen repairing buffer solution in a microwave oven, stopping heating for 5min with medium fire for 5min, turning to medium fire for 10min with medium fire, preventing excessive evaporation of the buffer solution, cutting into dry slices, naturally cooling, placing the slices in PBS, and shaking and washing for 3 times with 5min each time;
s16: adding a third primary antibody: dropwise adding the diluted third primary antibody on the slices, and flatly placing the slices in a wet box for incubation overnight at 4 ℃ in a dark place;
s17: adding a corresponding secondary HRP-labeled antibody: placing the slide in PBS, shaking on a decoloring shaking table to wash in the dark for 3 times, each time for 5min, dripping a second antibody covering tissue corresponding to the HRP mark into a ring after the slide is slightly dried, and incubating in the dark for 1h in a room temperature wet box;
s18: kit reagents were stained using multiple IF: placing the slide in PBS, and washing for 5min each time for 3 times; dripping the prepared multiple IF staining working solution into the ring, and incubating the inner chamber of the wet box for 30min in a warm and dark place; placing the slide in PBS, and washing for 5min each time for 2 times in a manner of shaking on a decoloring shaking table in a dark place;
s19: adding Cy5 working solution: after the slices are slightly dried, Cy5 working solution is dripped into the ring to cover the tissues, and the tissues are incubated in a room-temperature wet box for 30min in a dark place;
s20: DAPI counterstained nuclei: placing the slide in PBS, shaking and washing for 3 times on a decoloring shaking table for 5min each time, dripping DAPI dye liquor into the ring after the slide is slightly dried, and incubating for 10min at room temperature in a dark place;
s21: sealing: placing the slide in PBS, shaking and washing for 3 times on a decoloring shaking table, each time for 5min, slightly drying the slide, and sealing the slide by using an anti-fluorescence quenching sealing agent;
s22: microscopic examination: and taking a picture of the selected visual field under a microscope.
Preferably, in S1, reagent 1, reagent 2, the multiple IF staining working solution, the FITC working solution, the Cy3 working solution and the Cy5 working solution are mixed uniformly and protected from light for standby, and the multiple IF triple staining working solution is prepared according to the calculated amount of the sample.
Preferably, the pH value of the EDTA antigen repair buffer is 8.0, and the pH value of the PBS is 7.4.
Preferably, in S4, after incubation of the autofluorescence quencher B, the tissue is stained blue-black, which does not affect the results of the subsequent experiments.
Preferably, in S5, 5g of BSA is weighed and dissolved in 100ml of PBS to obtain a BSA solution with a mass/volume ratio of 5%.
Preferably, the addition of the first primary antibody, the second primary antibody and the third primary antibody prevents evaporation of the antibodies by adding a small amount of water to the wet cell.
Advantageous effects
The kit adopts a TSA signal amplification technology, the principle of the kit is similar to that of common immunohistochemistry, a primary antibody is combined with an antigen, an HRP-labeled secondary antibody is incubated, a fluorescein substrate of a system is added in an HRP catalysis mode to generate an activated fluorescein substrate, the activated substrate can be covalently combined with tyrosine on the antigen, so that fluorescein is stably covalently combined on a sample, then the non-covalently combined antibody is washed away by using heat restoration, then a second primary antibody is replaced for second round incubation, another fluorescein substrate is replaced, multiple labeling can be realized by repeating the steps, in addition, only one antibody is incubated in each round, and the non-covalently combined antibody is washed away after microwave treatment, so the source of the used antibody is not limited.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic diagram of the TSA of the present invention;
FIG. 2 is a view of a first embodiment of the present invention;
FIG. 3 is a microscopic E-Cadherin view of a mouse lung of the present invention;
FIG. 4 is a view of the invention under a microscope KLF 4;
FIG. 5 is a microscopic view of the present invention;
FIG. 6 is a view of a second embodiment of the present invention;
FIG. 7 is a microscopic BDNF view of rat brain according to the present invention;
FIG. 8 is a microscopic Iba1 view of the present invention;
FIG. 9 is a Neun view under a microscope of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the description of the present invention, it is to be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", and the like, indicate orientations and positional relationships based on those shown in the drawings, and are used only for convenience of description and simplicity of description, and do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be considered as limiting the present invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, features defined as "first", "second", may explicitly or implicitly include one or more of the described features. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise. Furthermore, the terms "mounted," "connected," and "connected" are to be construed broadly and may, for example, be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.
Referring to fig. 1 to 9, the present invention provides a technical solution:
a paraffin section immunofluorescence multiple staining kit comprises a reagent A, a reagent B, a diluent, a FITC reagent, a Cy3 reagent and a Cy5 reagent;
the reagent A is mainly Biotin-Tyramide;
the reagent B is 30% of H2O2；
The diluent is a phosphate buffer solution with the pH value of 7.4;
the FITC reagent is Streptavidin-FITC;
the Cy3 reagent is Streptavidin-Cy 3;
the Cy5 reagent is Streptavidin-Cy 5.
A method for using a paraffin section immunofluorescence multiple staining kit comprises the following steps:
s1: reagent preparation, comprising the steps of:
s1-1: dissolving 1ul of reagent A in 500ul of diluent to serve as a reagent 1, and uniformly mixing and keeping out of the sun for later use;
s1-2: dissolving 1ul of reagent B in 10ul of diluent to serve as a reagent 2, and uniformly mixing and keeping out of the sun for later use;
s1-3: dissolving 1ul of the reagent 2 in 100ul of the reagent 1 to prepare multiple IF dyeing working solution, uniformly mixing and keeping out of the sun for standby, and preparing the multiple IF triple-dyeing working solution for use according to the calculated amount of a sample;
s1-4: dissolving 1ul FITC reagent in 1ml diluent to obtain FITC working solution, mixing well and keeping out of the sun for later use;
s1-5: dissolving 1ul of Cy3 reagent in 1ml of diluent to prepare Cy3 working solution, and uniformly mixing and keeping out of the sun for later use;
s1-6: dissolving 1ul of Cy5 reagent in 1ml of diluent to prepare Cy5 working solution, and uniformly mixing and keeping out of the sun for later use;
s2: paraffin section dewaxing to water: sequentially placing the slices in xylene I15 min, xylene II 15min, anhydrous ethanol I5 min, anhydrous ethanol II 5min, 85% ethanol 5min and 75% ethanol 5min, and washing with distilled water;
s3: antigen retrieval: placing the tissue slices in a repairing box filled with EDTA antigen repairing buffer solution, performing antigen repairing in a steamer, timing for 30min after the temperature reaches 95 ℃, placing the slides in PBS after natural cooling, and shaking and washing the slides on a decoloring shaking table for 3 times, 5min each time;
s4: circle drawing autofluorescence quenching: after the section is slightly dried, a circle is drawn around the tissue by a histochemical pen to prevent the antibody from flowing away, an autofluorescence quencher A is added into the circle, the circle is incubated for 15min at room temperature and then washed by running water for 10min, an autofluorescence quencher B is added into the circle, the circle is incubated for 10min at room temperature and then washed by running water for 10min, after the autofluorescence quencher B is incubated, the tissue is dyed into blue black, and the subsequent experimental result cannot be influenced;
s5: serum blocking, namely dropwise adding 5% BSA in a circle, incubating at room temperature for 30min, weighing 5g BSA, and dissolving in 100ml PBS to obtain a BSA solution with the mass-volume ratio of 5%;
s6: adding a first primary antibody: removing the confining liquid, dripping the first primary antibody into the ring, incubating overnight at 4 ℃ in a wet box, and adding a small amount of water into the wet box to prevent the antibody from evaporating;
s7: adding a corresponding secondary HRP-labeled antibody: placing the slide in PBS, shaking and washing for 3 times on a decoloring shaking table for 5min each time, dripping a second antibody labeled by corresponding HRP in a ring to cover the tissue after the slide is slightly dried, and incubating for 1h in a room temperature wet box;
s8: kit reagents were stained using multiple IF: placing the slide in PBS, and washing for 5min each time for 3 times; dripping the prepared multiple IF staining working solution into the ring, and incubating the inner chamber of the wet box for 30min in a warm and dark place; placing the slide in PBS, and washing for 5min each time for 2 times in a manner of shaking on a decoloring shaking table in a dark place;
s9: adding FITC working solution: after the section is slightly dried, FITC working solution is dripped into the ring to cover the tissue, and the tissue is incubated for 30min in a room temperature wet box in a dark place;
s10: microwave treatment to remove excess primary antibody: placing the tissue slices in a repairing box filled with EDTA antigen repairing buffer solution in a microwave oven, stopping heating for 5min with medium fire for 5min, turning to medium fire for 10min with medium fire, preventing excessive evaporation of the buffer solution, cutting into dry slices, naturally cooling, placing the slices in PBS, and shaking and washing for 3 times with 5min each time;
s11: adding a second primary antibody: dropwise adding the diluted second primary antibody on the slice, flatly placing the slice in a wet box, incubating overnight at 4 ℃ in a dark place, and adding a small amount of water in the wet box to prevent the antibody from evaporating;
s12: adding a corresponding secondary HRP-labeled antibody: placing the slide in PBS, shaking on a decoloring shaking table to wash in the dark for 3 times, each time for 5min, dripping a second antibody covering tissue corresponding to the HRP mark into a ring after the slide is slightly dried, and incubating in the dark for 1h in a room temperature wet box;
s13: kit reagents were stained using multiple IF: placing the slide in PBS, and washing for 5min each time for 3 times; dripping the prepared multiple IF staining working solution into the ring, and incubating the inner chamber of the wet box for 30min in a warm and dark place; placing the slide in PBS, and washing for 5min each time for 2 times in a manner of shaking on a decoloring shaking table in a dark place;
s14: adding Cy3 working solution: after the slices are slightly dried, Cy3 working solution is dripped into the ring to cover the tissues, and the tissues are incubated in a room-temperature wet box for 30min in a dark place;
s15: microwave treatment to remove excess primary antibody: placing the tissue slices in a repairing box filled with EDTA antigen repairing buffer solution in a microwave oven, stopping heating for 5min with medium fire for 5min, turning to medium fire for 10min with medium fire, preventing excessive evaporation of the buffer solution, cutting into dry slices, naturally cooling, placing the slices in PBS, and shaking and washing for 3 times with 5min each time;
s16: adding a third primary antibody: dropwise adding the diluted third primary antibody on the slices, flatly placing the slices in a wet box, incubating overnight at 4 ℃ in a dark place, and adding a small amount of water in the wet box to prevent the antibodies from evaporating;
s17: adding a corresponding secondary HRP-labeled antibody: placing the slide in PBS, shaking on a decoloring shaking table to wash in the dark for 3 times, each time for 5min, dripping a second antibody covering tissue corresponding to the HRP mark into a ring after the slide is slightly dried, and incubating in the dark for 1h in a room temperature wet box;
s18: kit reagents were stained using multiple IF: placing the slide in PBS, and washing for 5min each time for 3 times; dripping the prepared multiple IF staining working solution into the ring, and incubating the inner chamber of the wet box for 30min in a warm and dark place; placing the slide in PBS, and washing for 5min each time for 2 times in a manner of shaking on a decoloring shaking table in a dark place;
s19: adding Cy5 working solution: after the slices are slightly dried, Cy5 working solution is dripped into the ring to cover the tissues, and the tissues are incubated in a room-temperature wet box for 30min in a dark place;
s20: DAPI counterstained nuclei: placing the slide in PBS, shaking and washing for 3 times on a decoloring shaking table for 5min each time, dripping DAPI dye liquor into the ring after the slide is slightly dried, and incubating for 10min at room temperature in a dark place;
s21: sealing: placing the slide in PBS, shaking and washing for 3 times on a decoloring shaking table, each time for 5min, slightly drying the slide, and sealing the slide by using an anti-fluorescence quenching sealing agent;
s22: microscopic examination: and taking a picture of the selected visual field under a microscope.
Preferably, in S1, reagent 1, reagent 2, the multiple IF staining working solution, the FITC working solution, the Cy3 working solution and the Cy5 working solution are mixed uniformly and protected from light for standby, and the multiple IF triple staining working solution is prepared according to the calculated amount of the sample.
The pH value of the EDTA antigen retrieval buffer solution is 8.0, and the pH value of the PBS is 7.4.
As a first embodiment of the present invention: an application method of a paraffin section immunofluorescence multiple staining kit adopts E-Cadherin, KLF4 and Vimentin immunofluorescence homologous triple staining of mouse lungs, and comprises the following steps:
s1: reagent preparation, comprising the steps of:
s1-1: dissolving 1ul of reagent A in 500ul of diluent to serve as a reagent 1, and uniformly mixing and keeping out of the sun for later use;
s1-2: dissolving 1ul of reagent B in 10ul of diluent to serve as a reagent 2, and uniformly mixing and keeping out of the sun for later use;
s1-3: dissolving 1ul of the reagent 2 in 100ul of the reagent 1 to prepare multiple IF dyeing working solution, uniformly mixing and keeping out of the sun for standby, and preparing the multiple IF triple-dyeing working solution for use according to the calculated amount of a sample;
s1-4: dissolving 1ul FITC reagent in 1ml diluent to obtain FITC working solution, mixing well and keeping out of the sun for later use;
s1-5: dissolving 1ul of Cy3 reagent in 1ml of diluent to prepare Cy3 working solution, and uniformly mixing and keeping out of the sun for later use;
s1-6: dissolving 1ul of Cy5 reagent in 1ml of diluent to prepare Cy5 working solution, and uniformly mixing and keeping out of the sun for later use;
s2: paraffin section dewaxing to water: sequentially placing the slices in xylene I15 min, xylene II 15min, anhydrous ethanol I5 min, anhydrous ethanol II 5min, 85% ethanol 5min and 75% ethanol 5min, and washing with distilled water;
s3: antigen retrieval: placing the tissue slices in a repairing box filled with EDTA antigen repairing buffer solution, performing antigen repairing in a steamer, timing for 30min after the temperature reaches 95 ℃, placing the slides in PBS after natural cooling, and shaking and washing the slides on a decoloring shaking table for 3 times, 5min each time;
s4: circle drawing autofluorescence quenching: after the section is slightly dried, a circle is drawn around the tissue by a histochemical pen to prevent the antibody from flowing away, an autofluorescence quencher A is added into the circle, the circle is incubated for 15min at room temperature and then washed by running water for 10min, an autofluorescence quencher B is added into the circle, the circle is incubated for 10min at room temperature and then washed by running water for 10min, after the autofluorescence quencher B is incubated, the tissue is dyed into blue black, and the subsequent experimental result cannot be influenced;
s5: serum blocking, namely dropwise adding 5% BSA in a circle, incubating at room temperature for 30min, weighing 5g BSA, and dissolving in 100ml PBS to obtain a BSA solution with the mass-volume ratio of 5%;
s6: adding E-Cadherin primary antibody: removing the confining liquid, dripping E-Cadherin primary antibody into the ring, incubating overnight at 4 ℃ in a wet box, and adding a small amount of water in the wet box to prevent the antibody from evaporating;
s7: adding a corresponding secondary HRP-labeled antibody: placing the slide in PBS, shaking and washing for 3 times on a decoloring shaking table, each time for 5min, dripping Goatanti-Rabbit IgG H & L (HRP) secondary antibody in a ring after the slide is slightly dried, covering tissues, and incubating for 1H in a room-temperature wet box;
s8: kit reagents were stained using multiple IF: placing the slide in PBS, and washing for 5min each time for 3 times; dripping the prepared multiple IF staining working solution into the ring, and incubating the inner chamber of the wet box for 30min in a warm and dark place; placing the slide in PBS, and washing for 5min each time for 2 times in a manner of shaking on a decoloring shaking table in a dark place;
s9: adding FITC working solution: after the section is slightly dried, FITC working solution is dripped into the ring to cover the tissue, and the tissue is incubated for 30min in a room temperature wet box in a dark place;
s10: microwave treatment to remove excess primary antibody: placing the tissue slices in a repairing box filled with EDTA antigen repairing buffer solution in a microwave oven, stopping heating for 5min with medium fire for 5min, turning to medium fire for 10min with medium fire, preventing excessive evaporation of the buffer solution, cutting into dry slices, naturally cooling, placing the slices in PBS, and shaking and washing for 3 times with 5min each time;
s11: addition of KLF4 primary antibody: dripping diluted KLF4 primary antibody on the slice, flatly placing the slice in a wet box, incubating overnight at 4 ℃ in the dark, and adding a small amount of water in the wet box to prevent the antibody from evaporating;
s12: adding a corresponding secondary HRP-labeled antibody: placing the slide in PBS, shaking on a decoloring shaking table to wash in the dark for 3 times, each time for 5min, dripping Goatanti-Rabbit IgGH & L (HRP) secondary antibody covering tissues into a ring after the slide is slightly dried, and incubating in the dark for 1h in a room temperature wet box;
s13: kit reagents were stained using multiple IF: placing the slide in PBS, and washing for 5min each time for 3 times; dripping the prepared multiple IF staining working solution into the ring, and incubating the inner chamber of the wet box for 30min in a warm and dark place; placing the slide in PBS, and washing for 5min each time for 2 times in a manner of shaking on a decoloring shaking table in a dark place;
s14: adding Cy3 working solution: after the slices are slightly dried, Cy3 working solution is dripped into the ring to cover the tissues, and the tissues are incubated in a room-temperature wet box for 30min in a dark place;
s15: microwave treatment to remove excess primary antibody: placing the tissue slices in a repairing box filled with EDTA antigen repairing buffer solution in a microwave oven, stopping heating for 5min with medium fire for 5min, turning to medium fire for 10min with medium fire, preventing excessive evaporation of the buffer solution, cutting into dry slices, naturally cooling, placing the slices in PBS, and shaking and washing for 3 times with 5min each time;
s16: adding Vimentin primary antibody: dropwise adding the diluted Vimentin primary antibody on the slices, flatly placing the slices in a wet box, incubating overnight at 4 ℃ in a dark place, and adding a small amount of water in the wet box to prevent the antibody from evaporating;
s17: adding a corresponding secondary HRP-labeled antibody: placing the slide in PBS, shaking on a decoloring shaking table to wash in the dark for 3 times, each time for 5min, dripping Goatanti-Rabbit IgGH & L (HRP) secondary antibody covering tissues into a ring after the slide is slightly dried, and incubating in the dark for 1h in a room temperature wet box;
s18: kit reagents were stained using multiple IF: placing the slide in PBS, and washing for 5min each time for 3 times; dripping the prepared multiple IF staining working solution into the ring, and incubating the inner chamber of the wet box for 30min in a warm and dark place; placing the slide in PBS, and washing for 5min each time for 2 times in a manner of shaking on a decoloring shaking table in a dark place;
s19: adding Cy5 working solution: after the slices are slightly dried, Cy5 working solution is dripped into the ring to cover the tissues, and the tissues are incubated in a room-temperature wet box for 30min in a dark place;
s20: DAPI counterstained nuclei: placing the slide in PBS, shaking and washing for 3 times on a decoloring shaking table for 5min each time, dripping DAPI dye liquor into the ring after the slide is slightly dried, and incubating for 10min at room temperature in a dark place;
s21: sealing: placing the slide in PBS, shaking and washing for 3 times on a decoloring shaking table, each time for 5min, slightly drying the slide, and sealing the slide by using an anti-fluorescence quenching sealing agent;
s22: microscopic examination: selecting a visual field under a microscope for photographing;
as shown in the results of E-Cadherin, KLF4 and Vimentin immunofluorescence homologous triple-staining of mouse lung in FIGS. 2 to 5, the kit can mark multiple antigens on the same paraffin section, and has no limitation on one anti-source species.
As a second embodiment of the present invention: a method for using a paraffin section immunofluorescence multiple staining kit adopts rat brain BDNF, Iba1 and Neun immunofluorescence homologous triple staining, and comprises the following steps:
s1: reagent preparation, comprising the steps of:
s1-1: dissolving 1ul of reagent A in 500ul of diluent to serve as a reagent 1, and uniformly mixing and keeping out of the sun for later use;
s1-2: dissolving 1ul of reagent B in 10ul of diluent to serve as a reagent 2, and uniformly mixing and keeping out of the sun for later use;
s1-3: dissolving 1ul of the reagent 2 in 100ul of the reagent 1 to prepare multiple IF dyeing working solution, uniformly mixing and keeping out of the sun for standby, and preparing the multiple IF triple-dyeing working solution for use according to the calculated amount of a sample;
s1-4: dissolving 1ul FITC reagent in 1ml diluent to obtain FITC working solution, mixing well and keeping out of the sun for later use;
s1-5: dissolving 1ul of Cy3 reagent in 1ml of diluent to prepare Cy3 working solution, and uniformly mixing and keeping out of the sun for later use;
s1-6: dissolving 1ul of Cy5 reagent in 1ml of diluent to prepare Cy5 working solution, and uniformly mixing and keeping out of the sun for later use;
s2: paraffin section dewaxing to water: sequentially placing the slices in xylene I15 min, xylene II 15min, anhydrous ethanol I5 min, anhydrous ethanol II 5min, 85% ethanol 5min and 75% ethanol 5min, and washing with distilled water;
s3: antigen retrieval: placing the tissue slices in a repairing box filled with EDTA antigen repairing buffer solution, performing antigen repairing in a steamer, timing for 30min after the temperature reaches 95 ℃, placing the slides in PBS after natural cooling, and shaking and washing the slides on a decoloring shaking table for 3 times, 5min each time;
s4: circle drawing autofluorescence quenching: after the section is slightly dried, a circle is drawn around the tissue by a histochemical pen to prevent the antibody from flowing away, an autofluorescence quencher A is added into the circle, the circle is incubated for 15min at room temperature and then washed by running water for 10min, an autofluorescence quencher B is added into the circle, the circle is incubated for 10min at room temperature and then washed by running water for 10min, after the autofluorescence quencher B is incubated, the tissue is dyed into blue black, and the subsequent experimental result cannot be influenced;
s5: serum blocking, namely dropwise adding 5% BSA in a circle, incubating at room temperature for 30min, weighing 5g BSA, and dissolving in 100ml PBS to obtain a BSA solution with the mass-volume ratio of 5%;
s6: adding BDNF primary antibody: removing the confining liquid, dripping BDNF primary antibody into the ring, incubating overnight at 4 ℃ in a wet box, and adding a small amount of water into the wet box to prevent the antibody from evaporating;
s7: adding a corresponding secondary HRP-labeled antibody: placing the slide in PBS, shaking and washing for 3 times on a decoloring shaking table, each time for 5min, dripping Goatanti-Rabbit IgG H & L (HRP) secondary antibody in a ring after the slide is slightly dried, covering tissues, and incubating for 1H in a room-temperature wet box;
s8: kit reagents were stained using multiple IF: placing the slide in PBS, and washing for 5min each time for 3 times; dripping the prepared multiple IF staining working solution into the ring, and incubating the inner chamber of the wet box for 30min in a warm and dark place; placing the slide in PBS, and washing for 5min each time for 2 times in a manner of shaking on a decoloring shaking table in a dark place;
s9: adding FITC working solution: after the section is slightly dried, FITC working solution is dripped into the ring to cover the tissue, and the tissue is incubated for 30min in a room temperature wet box in a dark place;
s10: microwave treatment to remove excess primary antibody: placing the tissue slices in a repairing box filled with EDTA antigen repairing buffer solution in a microwave oven, stopping heating for 5min with medium fire for 5min, turning to medium fire for 10min with medium fire, preventing excessive evaporation of the buffer solution, cutting into dry slices, naturally cooling, placing the slices in PBS, and shaking and washing for 3 times with 5min each time;
s11: adding Iba1 primary antibody: dropwise adding the diluted Iba1 primary antibody on the slices, flatly placing the slices in a wet box, incubating overnight at 4 ℃ in a dark place, and adding a small amount of water in the wet box to prevent the antibody from evaporating;
s12: adding a corresponding secondary HRP-labeled antibody: placing the slide in PBS, shaking on a decoloring shaking table to wash in the dark for 3 times, each time for 5min, dripping Goatanti-Rabbit IgG H & L (HRP) secondary antibody covering tissues into a ring after the slide is slightly dried, and incubating in the dark for 1H in a room temperature wet box;
s13: kit reagents were stained using multiple IF: placing the slide in PBS, and washing for 5min each time for 3 times; dripping the prepared multiple IF staining working solution into the ring, and incubating the inner chamber of the wet box for 30min in a warm and dark place; placing the slide in PBS, and washing for 5min each time for 2 times in a manner of shaking on a decoloring shaking table in a dark place;
s14: adding Cy3 working solution: after the slices are slightly dried, Cy3 working solution is dripped into the ring to cover the tissues, and the tissues are incubated in a room-temperature wet box for 30min in a dark place;
s15: microwave treatment to remove excess primary antibody: placing the tissue slices in a repairing box filled with EDTA antigen repairing buffer solution in a microwave oven, stopping heating for 5min with medium fire for 5min, turning to medium fire for 10min with medium fire, preventing excessive evaporation of the buffer solution, cutting into dry slices, naturally cooling, placing the slices in PBS, and shaking and washing for 3 times with 5min each time;
s16: adding a Neun primary antibody: dropwise adding the diluted Neun primary antibody on the slices, flatly placing the slices in a wet box, incubating overnight at 4 ℃ in a dark place, and adding a small amount of water in the wet box to prevent the antibody from evaporating;
s17: adding a corresponding secondary HRP-labeled antibody: placing the slide in PBS, shaking on a decoloring shaking table to wash in the dark for 3 times, each time for 5min, dripping Goatanti-Rabbit IgG H & L (HRP) secondary antibody covering tissues into a ring after the slide is slightly dried, and incubating in the dark for 1H in a room temperature wet box;
s18: kit reagents were stained using multiple IF: placing the slide in PBS, and washing for 5min each time for 3 times; dripping the prepared multiple IF staining working solution into the ring, and incubating the inner chamber of the wet box for 30min in a warm and dark place; placing the slide in PBS, and washing for 5min each time for 2 times in a manner of shaking on a decoloring shaking table in a dark place;
s19: adding Cy5 working solution: after the slices are slightly dried, Cy5 working solution is dripped into the ring to cover the tissues, and the tissues are incubated in a room-temperature wet box for 30min in a dark place;
s20: DAPI counterstained nuclei: placing the slide in PBS, shaking and washing for 3 times on a decoloring shaking table for 5min each time, dripping DAPI dye liquor into the ring after the slide is slightly dried, and incubating for 10min at room temperature in a dark place;
s21: sealing: placing the slide in PBS, shaking and washing for 3 times on a decoloring shaking table, each time for 5min, slightly drying the slide, and sealing the slide by using an anti-fluorescence quenching sealing agent;
s22: microscopic examination: selecting a visual field under a microscope for photographing;
from the results of the rat brain BDNF, Iba1 and Neun immunofluorescence homologous triple staining in fig. 6 to 9, it can be seen that the kit can mark a plurality of antigens on the same paraffin section, and there is no limitation to one antigen source species.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (7)
1. A paraffin section immunofluorescence multiple staining kit comprises a reagent A, a reagent B, a diluent, a FITC reagent, a Cy3 reagent and a Cy5 reagent;
the reagent A is mainly Biotin-Tyramide;
the reagent B is 30% of H2O2；
The diluent is a phosphate buffer solution with the pH value of 7.4;
the FITC reagent is Streptavidin-FITC;
the Cy3 reagent is Streptavidin-Cy 3;
the Cy5 reagent is Streptavidin-Cy 5.
2. The use method of the paraffin section immunofluorescence multiple staining kit is characterized by comprising the following steps:
s1: reagent preparation, comprising the steps of:
s1-1: dissolving 1ul of reagent A in 500ul of diluent to obtain a reagent 1;
s1-2: dissolving 1ul of reagent B in 10ul of diluent to obtain a reagent 2;
s1-3: dissolving 1ul of the reagent 2 in 100ul of the reagent 1 to prepare a multiple IF staining working solution;
s1-4: dissolving 1ul of FITC reagent in 1ml of diluent to prepare FITC working solution;
s1-5: dissolving 1ul of Cy3 reagent in 1ml of diluent to prepare Cy3 working solution;
s1-6: dissolving 1ul of Cy5 reagent in 1ml of diluent to prepare Cy5 working solution;
s2: paraffin section dewaxing to water: sequentially placing the slices in xylene I15 min, xylene II 15min, anhydrous ethanol I5 min, anhydrous ethanol II 5min, 85% ethanol 5min and 75% ethanol 5min, and washing with distilled water;
s3: antigen retrieval: placing the tissue slices in a repairing box filled with EDTA antigen repairing buffer solution, performing antigen repairing in a steamer, timing for 30min after the temperature reaches 95 ℃, placing the slides in PBS after natural cooling, and shaking and washing the slides on a decoloring shaking table for 3 times, 5min each time;
s4: circle drawing autofluorescence quenching: after the section is slightly dried, a circle is drawn around the tissue by a tissue pen to prevent the antibody from flowing away, an autofluorescence quencher A is added into the circle, the circle is incubated for 15min at room temperature, then washed by flowing water for 10min, an autofluorescence quencher B is added into the circle, the circle is incubated for 10min at room temperature, and then washed by flowing water for 10 min;
s5: sealing serum, namely dropwise adding 5% BSA in a circle, and incubating for 30min at room temperature;
s6: adding a first primary antibody: removing the confining liquid, dripping the first primary antibody into the ring, and incubating overnight at 4 ℃ in a wet box;
s7: adding a corresponding secondary HRP-labeled antibody: placing the slide in PBS, shaking and washing for 3 times on a decoloring shaking table for 5min each time, dripping a second antibody labeled by corresponding HRP in a ring to cover the tissue after the slide is slightly dried, and incubating for 1h in a room temperature wet box;
s8: kit reagents were stained using multiple IF: placing the slide in PBS, and washing for 5min each time for 3 times; dripping the prepared multiple IF staining working solution into the ring, and incubating the inner chamber of the wet box for 30min in a warm and dark place; placing the slide in PBS, and washing for 5min each time for 2 times in a manner of shaking on a decoloring shaking table in a dark place;
s9: adding FITC working solution: after the section is slightly dried, FITC working solution is dripped into the ring to cover the tissue, and the tissue is incubated for 30min in a room temperature wet box in a dark place;
s10: microwave treatment to remove excess primary antibody: placing the tissue slices in a repairing box filled with EDTA antigen repairing buffer solution in a microwave oven, stopping heating for 5min with medium fire for 5min, turning to medium fire for 10min with medium fire, preventing excessive evaporation of the buffer solution, cutting into dry slices, naturally cooling, placing the slices in PBS, and shaking and washing for 3 times with 5min each time;
s11: adding a second primary antibody: dropwise adding the diluted second primary antibody on the slices, and flatly placing the slices in a wet box for incubation overnight at 4 ℃ in a dark place;
s12: adding a corresponding secondary HRP-labeled antibody: placing the slide in PBS, shaking on a decoloring shaking table to wash in the dark for 3 times, each time for 5min, dripping a second antibody covering tissue corresponding to the HRP mark into a ring after the slide is slightly dried, and incubating in the dark for 1h in a room temperature wet box;
s13: kit reagents were stained using multiple IF: placing the slide in PBS, and washing for 5min each time for 3 times; dripping the prepared multiple IF staining working solution into the ring, and incubating the inner chamber of the wet box for 30min in a warm and dark place; placing the slide in PBS, and washing for 5min each time for 2 times in a manner of shaking on a decoloring shaking table in a dark place;
s14: adding Cy3 working solution: after the slices are slightly dried, Cy3 working solution is dripped into the ring to cover the tissues, and the tissues are incubated in a room-temperature wet box for 30min in a dark place;
s15: microwave treatment to remove excess primary antibody: placing the tissue slices in a repairing box filled with EDTA antigen repairing buffer solution in a microwave oven, stopping heating for 5min with medium fire for 5min, turning to medium fire for 10min with medium fire, preventing excessive evaporation of the buffer solution, cutting into dry slices, naturally cooling, placing the slices in PBS, and shaking and washing for 3 times with 5min each time;
s16: adding a third primary antibody: dropwise adding the diluted third primary antibody on the slices, and flatly placing the slices in a wet box for incubation overnight at 4 ℃ in a dark place;
s17: adding a corresponding secondary HRP-labeled antibody: placing the slide in PBS, shaking on a decoloring shaking table to wash in the dark for 3 times, each time for 5min, dripping a second antibody covering tissue corresponding to the HRP mark into a ring after the slide is slightly dried, and incubating in the dark for 1h in a room temperature wet box;
s18: kit reagents were stained using multiple IF: placing the slide in PBS, and washing for 5min each time for 3 times; dripping the prepared multiple IF staining working solution into the ring, and incubating the inner chamber of the wet box for 30min in a warm and dark place; placing the slide in PBS, and washing for 5min each time for 2 times in a manner of shaking on a decoloring shaking table in a dark place;
s19: adding Cy5 working solution: after the slices are slightly dried, Cy5 working solution is dripped into the ring to cover the tissues, and the tissues are incubated in a room-temperature wet box for 30min in a dark place;
s20: DAPI counterstained nuclei: placing the slide in PBS, shaking and washing for 3 times on a decoloring shaking table for 5min each time, dripping DAPI dye liquor into the ring after the slide is slightly dried, and incubating for 10min at room temperature in a dark place;
s21: sealing: placing the slide in PBS, shaking and washing for 3 times on a decoloring shaking table, each time for 5min, slightly drying the slide, and sealing the slide by using an anti-fluorescence quenching sealing agent;
s22: microscopic examination: and taking a picture of the selected visual field under a microscope.
3. The use method of the paraffin section immunofluorescence multiple staining kit according to claim 2, wherein in S1, the reagent 1, the reagent 2, the multiple IF staining working solution, the FITC working solution, the Cy3 working solution and the Cy5 working solution are uniformly mixed and protected from light for standby, and the multiple IF triple staining working solution is prepared according to the calculated amount of the sample.
4. The use method of the paraffin section immunofluorescence multiple staining kit according to claim 2, wherein the pH value of the EDTA antigen retrieval buffer is 8.0, and the pH value of the PBS is 7.4.
5. The method for using the paraffin section immunofluorescence multiple staining kit according to claim 2, wherein, in S4, after the autofluorescence quencher B is incubated, the tissue is stained with blue black, and the result does not affect the results of subsequent experiments.
6. The method for using the paraffin section immunofluorescence multiple staining kit of claim 2, wherein in S5, 5g BSA is weighed and dissolved in 100ml PBS, and a BSA solution with a mass volume ratio of 5% is obtained.
7. The method for using the paraffin section immunofluorescence multiple staining kit according to claim 2, wherein the first primary antibody, the second primary antibody and the third primary antibody are added, and a small amount of water is added in a wet box to prevent the antibodies from evaporating.
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