CN111307556A - Method for performing immunofluorescence staining after HE section fades - Google Patents
Method for performing immunofluorescence staining after HE section fades Download PDFInfo
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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Abstract
The invention discloses a method for performing immunofluorescence staining after HE sections fade, which comprises the following steps: soaking the slices subjected to HE staining in xylene, and removing cover glass and neutral gum; hydrating with gradient alcohol for fading, and soaking in 1% ethanol hydrochloride differentiation solution prepared with 75% alcohol for 1 min; after air drying, putting into 1% hydrochloric acid alcohol differentiation solution for 1min, and repeating the step S3 for several times until the color of the slice is completely faded; slowly flushing with tap water and then performing high-pressure repair; and (4) carrying out immunofluorescence staining on the section subjected to high-pressure repair. The method has the beneficial effects that the comparison under the same antigen retrieval method shows that the antigen expression intensity of the section dyed by the HE faded is equivalent to that of the section directly dyed by the blank sheet, which indicates that the INS antigen is well preserved by the method.
Description
Technical Field
The invention relates to the technical field of medical detection, in particular to a method for performing immunofluorescence staining after HE sections fade.
Background
The immunofluorescence detection technology has the advantages of strong specificity, high sensitivity, good practicability and the like [1], and is an important auxiliary technology in pathological detection. HE staining is a routine pathological detection staining method, and some meaningful morphological changes are found in HE staining. Sometimes, the confirmed diagnosis or further research of the lesion under HE staining needs immunofluorescence staining assistance, and at this time, the re-cut wax block is often selected, but the re-cut section is not the same tissue piece as the HE observation, the position needing to be observed is changed more or less, which is not beneficial to the precise comparative research with the original HE section, and even some tissue pieces are small, and the re-cut section can not cut the tissue section for immunofluorescence staining.
Disclosure of Invention
In view of the defects of the prior art, the invention discusses a dyeing method for performing immunofluorescence after HE section fading, and has important application value. The invention creates a simple and effective method for performing immunofluorescence staining after HE fading.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method of immunofluorescent staining of HE sections after discoloration, with HE stained sections, comprising the steps of:
s1 xylene soaking the sections with HE staining, removing coverslips and neutral gums;
s2 gradient alcohol hydration and color fading, soaking in 1% hydrochloric acid ethanol differentiation solution prepared with 75% alcohol for 1 min;
s3 air drying, adding 1% hydrochloric acid alcohol differentiation solution for 1min,
s4 repeating step S3 for several times until the color of the slice completely fades;
s5, slowly washing with tap water and then performing high-pressure repair;
s6 immunofluorescent staining of sections after high pressure repair.
Preferably, the high-pressure repairing is to soak the slices in the antigen repairing liquid, heat the slices in a pressure cooker, stop heating after 5min after air injection, and naturally cool the slices to room temperature.
Preferably, the immunofluorescent staining step in step S6 is to wash the section repaired by S6.1 high pressure with PBS 3 times for 5min each,
s6.2, sealing by fluorescent sealing liquid for 1h, incubating for one time at 4 ℃ in a refrigerator overnight, and rewarming for 37 ℃ for 60 min;
s6.3 washing with PBS for 3 times and 5min each time, incubating with secondary antibody at 37 deg.C for 60min, washing with PBS for 3 times and 5min each time, air drying, and sealing.
The method has the beneficial effects that the comparison under the same antigen retrieval method shows that the antigen expression intensity of the section dyed by the HE faded is equivalent to that of the section directly dyed by the blank sheet, which indicates that the INS antigen is well preserved by the method.
Drawings
FIG. 1 is a photograph of groups of control experiments A/B/C/D stained after fading according to the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings, and it should be noted that the following examples are provided to illustrate the detailed embodiments and specific operations based on the technical solutions of the present invention, but the scope of the present invention is not limited to the examples.
The invention relates to a method for performing immunofluorescence staining after HE sections fade, which comprises the following steps:
s1 xylene soaking the sections with HE staining, removing coverslips and neutral gums;
s2 gradient alcohol hydration and color fading, soaking in 1% hydrochloric acid ethanol differentiation solution prepared with 75% alcohol for 1 min;
s3 air drying, adding 1% hydrochloric acid alcohol differentiation solution for 1min,
s4 repeating step S3 for several times until the color of the slice completely fades;
s5, slowly washing with tap water and then performing high-pressure repair;
s6 immunofluorescent staining of sections after high pressure repair.
Preferably, the high-pressure repairing is to soak the slices in the antigen repairing liquid, heat the slices in a pressure cooker, stop heating after 5min after air injection, and naturally cool the slices to room temperature.
Preferably, the step of immunofluorescent staining in the step S6 is
S6.1 the section after high-pressure repair is washed for 5min for 3 times by PBS,
s6.2, sealing by fluorescent sealing liquid for 1h, incubating for one time at 4 ℃ in a refrigerator overnight, and rewarming for 37 ℃ for 60 min;
s6.3 washing with PBS for 3 times and 5min each time, incubating with secondary antibody at 37 deg.C for 60min, washing with PBS for 3 times and 5min each time, air drying, and sealing.
Examples
Experimental animal and experimental environment
[ general grade ] food crab monkeys 3, provided by Guangdong Chunsheng Biotechnology development Co., Ltd, were bred in the general environment animal facilities of Guangdong Chunsheng Biotechnology development Co., Ltd, and all experimental protocols involving animals were approved by animal care and ethical committee. Animal husbandry facilities strictly follow the regulations and requirements of the international committee for laboratory animal assessment and certification and management for daily animal husbandry care and management.
Instruments and reagents
The adopted instruments comprise a Leica TP1020 automatic dehydrator, a Leica EG1150H + C paraffin embedding machine, a Leica RM2255 full-automatic rotary slicer, a Leica ST5020+ SV5030 full-automatic dyeing and sealing integrated machine, and a Leica DM3000 upright fluorescence microscope.
The reagents used were 1% ethanolic hydrochloride differentiation solution, insulin antigen (Gene Tex.), PBS buffer (doctor De bioengineering Co., Ltd.), trypsin (Nanjing institute of bioengineering), immunostaining blocking solution (Biyuntian Biotechnology Co., Ltd.), fluorescent secondary antibody (Invitrogen Life Technologies Co., Ltd.), xylene (Centijin Baishi chemical Co., Ltd.), absolute ethanol (Guangzhou Zhongnan chemical Co., Ltd.), 95% ethanol (Guangzhou Kaxiu trade Co., Ltd.), 1% alcohol-soluble eosin (Guangzhou Kaxiu trade Co., Ltd.), Harris hematoxylin (Guangzhou Kaxiu trade Co., Ltd.), and TO clearing agent (Guangzhou Zhongnan chemical instruments Co., Ltd.).
Experimental methods
Group of
Choose the wax stone sample after 3 normal blood sugar cynomolgus monkey pancreas conventionality dehydration embedding, drag for the piece with polylysine anticreep slide after the continuous section, divide into 4 groups, every group is 3, promptly: blank section high-pressure repair immunofluorescence group (group A), blank section trypsin repair immunofluorescence group (group B), HE section faded high-pressure repair immunofluorescence group (group C), HE section faded trypsin repair immunofluorescence group (group D).
HE dyeing method
Baking undyed blank slices, soaking in xylene I and xylene II for 20min respectively for dewaxing, and dehydrating with gradient alcohol to pure water for 7 min. And then performing Harris hematoxylin staining for 10min, then placing the stained hematoxylin into tap water for soaking for 1min, then performing 1% hydrochloric acid ethanol differentiation for 5s, and transferring the stained hematoxylin to the tap water to turn blue for 15 min. After the blue returning is finished, transferring TO 90% ethanol for soaking for 7min, then carrying out 1% alcohol-soluble eosin dyeing for 30s, transferring TO 95% ethanol I, 95% ethanol II and 95% ethanol III for respectively soaking for 10s after the eosin dyeing, then respectively transferring TO anhydrous ethanol I and anhydrous ethanol II for dehydration for 5min respectively, finally respectively soaking in TO transparent agent I, TO transparent agent II for 7min, and sealing.
Immunofluorescence staining method
Group A
Baking a blank slice without dyeing, dewaxing, washing with PBS 3 times and 5min each time after gradient alcohol hydration, repairing under high pressure (stopping heating after 5min after air injection of an autoclave, and naturally cooling to room temperature), washing with PBS 3 times and 5min each time, sealing with fluorescent sealing liquid for 1h, performing primary anti-incubation (overnight in a refrigerator at 4 ℃), rewarming (37 ℃, 60min), washing with PBS 3 times and 5min each time, incubating with secondary antibodies (37 ℃, 60min), washing with PBS 3 times and 5min each time, drying in the air, and sealing.
Group B
In comparison to group A, the antigen was repaired with 1.25% trypsin (37 ℃, 30min), without high pressure repair, and the rest was the same as group A.
Group C
Soaking the slices with xylene after HE dyeing, removing cover glass and neutral gum, performing gradient alcohol hydration and color fading, soaking in 1% hydrochloric acid ethanol differentiation solution prepared by 75% alcohol for 1min, air drying, adding 1% hydrochloric acid alcohol differentiation solution lmin, repeating for several times until the color completely fades, and slowly washing with tap water. Then, the cells were washed 3 times with PBS for 5min each, and subjected to immunofluorescence staining in the same manner as in group A, such as by high pressure repair.
Group D
In comparison to group C, the antigen was repaired with 1.25% trypsin (37 ℃, 30min), without high pressure repair, and the rest was the same as for group C.
As shown in figure 1, the HE observes that the islets are different in size and distributed dispersedly, the islet cells are distributed in a rope shape, the cytoplasm is lightly stained, the islets β cells with INS positive fluorescence expression can be seen in each group of islets, after the treatment by the method, part of tissues of A, C groups of slices are broken and shed, the slices of B, D groups of slices are not broken and shed, and further, the areas of the C group and other groups are comprehensively compared, the positive cells of the C group and the A group are bright and clear, and the antigen expression intensity and the staining effect are good.
Various corresponding changes and modifications can be made by those skilled in the art based on the above technical solutions and concepts, and all such changes and modifications should be included in the protection scope of the present invention.
Claims (3)
- A method of immunofluorescent staining of HE sections after discoloration, with HE stained sections, characterized in that the method comprises the steps of:s1 xylene soaking the sections with HE staining, removing coverslips and neutral gums;s2 gradient alcohol hydration and color fading, soaking in 1% hydrochloric acid ethanol differentiation solution prepared with 75% alcohol for 1 min;s3 air drying, adding 1% hydrochloric acid alcohol differentiation solution for 1min,s4 repeating step S3 for several times until the color of the slice completely fades;s5, slowly washing with tap water and then performing high-pressure repair;s6 immunofluorescent staining of sections after high pressure repair.
- 2. The method for immunofluorescent staining after fading of HE section according to claim 1, wherein the high pressure repairing is carried out by immersing the section in an antigen repairing solution, heating the section in an autoclave, stopping heating 5min after air injection, and naturally cooling the section to room temperature.
- 3. The method for immunofluorescent staining of HE sections following discoloration according to claim 1, wherein the step of immunofluorescent staining in step S6 isS6.1 the section after high-pressure repair is washed for 5min for 3 times by PBS,s6.2, sealing by fluorescent sealing liquid for 1h, incubating for one time at 4 ℃ in a refrigerator overnight, and rewarming for 37 ℃ for 60 min;s6.3 washing with PBS for 3 times and 5min each time, incubating with secondary antibody at 37 deg.C for 60min, washing with PBS for 3 times and 5min each time, air drying, and sealing.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112798379A (en) * | 2021-01-29 | 2021-05-14 | 四川大学华西医院 | Immunofluorescence mounting solution remover and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101923019A (en) * | 2010-07-27 | 2010-12-22 | 华中科技大学 | Method for restoring color of faded pathological HE section |
| CN107643396A (en) * | 2017-09-15 | 2018-01-30 | 如皋福大工程技术研究院有限公司 | A kind of SABC antigen retrieval method |
-
2020
- 2020-03-10 CN CN202010159650.6A patent/CN111307556A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101923019A (en) * | 2010-07-27 | 2010-12-22 | 华中科技大学 | Method for restoring color of faded pathological HE section |
| CN107643396A (en) * | 2017-09-15 | 2018-01-30 | 如皋福大工程技术研究院有限公司 | A kind of SABC antigen retrieval method |
Non-Patent Citations (2)
| Title |
|---|
| 李丽 等: "HE染色切片褪色后再进行免疫组化染色方法的比较", 《数理医药学杂志》 * |
| 范瑾瑾 等: "石蜡切片多重免疫荧光染色优化条件探讨", 《中山大学学报(医学版)》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112798379A (en) * | 2021-01-29 | 2021-05-14 | 四川大学华西医院 | Immunofluorescence mounting solution remover and application thereof |
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Application publication date: 20200619 |