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Hengyuan Biotechnology | Immunohistochemical Experiment Strategy


Immunohistochemistry (IHC) is a technique that uses antibodies to detect proteins and other antigens in tissue sections, studying these antigens through localization, qualitative analysis, and relative quantification. The following is a complete guide to immunohistochemistry experiments summarized by Hengyuan Biotechnology for everyone.

1、 Experimental principle
Immunohistochemistry is based on antigen antibody reactions and chemical colorimetric principles. The antigen in tissue slices or cell specimens first binds to the primary antibody, then binds to the primary antibody through the secondary antibody, and finally visualizes the target antigen through a colorimetric system (such as DAB) or fluorescence system.

2、 Preparation before the experiment
Material preparation: tissue sections, antibodies, buffer solution, etc
Equipment inspection: Ensure that slicing machines, incubators, and other equipment are working properly
Workbench cleaning: Ensure no bacterial or foreign object contamination
Experimental plan: Develop standardized operating procedures and arrange experimental time reasonably

3、 Detailed operation steps

1. Sample processing
Organizational collection: After obtaining tissue through surgery or biopsy, immediately fix it in 10% neutral formalin (6-48 hours)
Dehydration embedding: Tissue is dehydrated by gradient alcohol and then embedded in paraffin after transparency with xylene
Slice preparation: Use a slicer to make 4-5 micron thick slices, bake at 60 ° C for 30-60 minutes

2. Dewaxing hydration
Soak in xylene solution (I) for 10 minutes
Soak in xylene solution (II) for 10 minutes
Soak in gradient alcohol (100%, 95%, 80%, 70%) for 5 minutes each
Rinse with distilled water

3. Antigen repair
Thermal repair: commonly used pH 6.0 citrate buffer or pH 9.0 Tris/EDTA buffer, heated at 95-100 ° C for 20 minutes
Enzyme repair: Treatment with trypsin or proteinase K
Freezing slices can eliminate or reduce antigen repair steps

4. Block endogenous substances
Treat with 3% hydrogen peroxide solution for 10 minutes to block endogenous peroxidase
Normal serum (homologous to secondary antibody) was blocked for 20 minutes to reduce non-specific binding

5. Primary antibody incubation
Choose primary antibodies with high specificity and strong affinity
Dilute the primary antibody with PBS or TBS (commonly used ratio 1:100-1:500)
Add primary antibody dropwise to cover the tissue, overnight at 4 ° C or at room temperature for 1-2 hours
Wash with PBS 3 times, each time for 5 minutes

6. Secondary antibody incubation
Select secondary antibodies that match the species of primary antibody
Incubate at room temperature for 30-60 minutes
Wash with PBS 3 times, each time for 5 minutes

7. Color reaction
DAB color development: Control reaction time under microscope (usually 1-10 minutes)
Fluorescence color development: Avoid light operation, choose appropriate fluorescent secondary antibody

8. Re dyeing and sealing
Hematoxylin counterstaining of cell nuclei for 1-2 minutes
Gradient alcohol dehydration, transparent xylene
Neutral resin sealing or using a sealing machine

4、 Key precautions
Sample quality: Ensure fresh organization, timely and sufficient fixation
Antibody selection: Verify antibody specificity and optimize dilution ratio
Antigen repair: Select appropriate methods based on antigen characteristics (pH value, time)
Comparison setting: Positive and negative controls must be set up
Standardization of experiments: Maintain consistent operating conditions to avoid batch differences
Reagent storage: Antibodies should be packaged and stored at -20 ° C to avoid repeated freezing and thawing

5、 Common problem solving
High background staining: increase blocking time, optimize antibody concentration, and extend washing time
No signal: Check the antigen repair method, verify the effectiveness of the antibody, and extend the incubation time
Non specific staining: Replace the blocking serum and adjust the specificity of the primary antibody
Organizational detachment: use anti detachment slides, optimize baking time and temperature

By strictly following the above process and precautions, accurate and reliable immunohistochemical results can be obtained. For beginners, it is recommended to start with standardized reagent kits and gradually master various technical details. Shanghai Hengyuan Biotechnology Co., Ltd. has its own laboratory and a professional technical research and development team. It is committed to the research and sales of various biological reagents, cells, serum, ELISA kits, and provides free testing services for experiments. We also offer exquisite technical services. If you have any experimental questions, please feel free to consult us and we will provide you with professional one-on-one technical guidance.  

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