immunohistochemical staining

Summary

Immunohistochemical staining refers to the combination of fluorescent or colorable chemicals on antibodies to detect the presence of target antigens in cells or tissues by using the immunological principle of the specific binding reaction between antigens and antibodies, which can be used not only to measure the amount of antigen manifested, but also to observe the location of the antigen manifested. As long as the antibody binds to substances that are antigenic, including proteins, nucleic acids, polysaccharides, pathogens, etc., they can be detected.

Operation method

immunohistochemical staining

Materials and Instruments

Fresh Tissue
Xylene Alcohol H2O2 Antigen Repair Solution Sodium Citrate Buffer Paraffin Wax Formalin PBS DAB Hematoxylin Resin Acetone Perforation Solution Sodium Citrate APES
Thermostatic shaker Autoclave Plastic sectioning racks Temperature-resistant glass containers -80℃ Refrigerator Constant-cold freezing sectioning machine Slides

Move

I. Experimental steps for immunohistochemical staining of paraffin sections:1. Paraffin sections should be dewaxed to water: (paraffin sections should be placed at 60℃ for 1 hour before staining).(1) Xylene I and II, 10 minutes each.(2) Gradient Alcohol: 100%, 2 min® 95%, 2 min® 80%, 2 min® 70% 2 min.(3) Distilled water wash: 5 min, 2 times (on shaker).(2) Endogenous peroxidase sequestered with hydrogen peroxide: 3% H2O2, 10 minutes at room temperature (protected from light).3. Distilled water wash: 5 min, 2 times (on shaker).4. Antigen repair:Depending on the antigen to be detected, select the appropriate method.Attachment: Preparation of antigen repair solution (10 mM pH 6.0 sodium citrate buffer)(1) Preparation of reserve solution:Liquid A: trisodium citrate-2H2O 29.41g + distilled water 1000mlLiquid B: Trisodium citrate-2H2O 21g + Distilled water 1000ml(2) Preparation of working solution: 82ml of liquid A + 18ml of liquid B + 900ml of distilled water.Method of antigen repair:(1) Autoclave treatment technique: sodium citrate buffer (10 mM, PH 6.0), submerge slices, cover, boil in autoclave, vaporize for 3 minutes and then cool slowly (tap water can be used to rinse outside the autoclave to aid cooling).(2) Microwave processing technology: use plastic slicing frame, placed in plastic or temperature-resistant glass containers, sodium citrate buffer submerged slices, select medium-high or high-grade, 5 minutes; remove and replenish the preheated sodium citrate buffer; and then select medium-high or high-grade, 5 minutes (...). Optimal temperature is 92-95°C)(3) Enzymatic digestion: this is omitted.Notes on antigen repair:(1) The tissue must not be dry.(2) The choice of antigen repair method varies from antibody to antibody.(3) This method is mainly used for 10% formalin-fixed, paraffin-embedded tissues.(4) PBS buffer is required after antigen fixation until DAB color development.(5) PBS: 5 minutes, twice (on shaker).6. Normal serum closure: take out the sections from the vat, wipe off the water on the back of the section and the water around the tissue on the front side of the section (keep the tissue in a moist state), add normal goat or rabbit serum (homologous to the second antibody) to treat it, 37℃, 15 minutes.

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