Reagents required for immunohistochemistry
1) PBS buffer (pH 7.2 ~ 7.4): NaCl 137mmol / L, KCl 2.7mmol / L, Na2HPO4 4.3mmol / L, KH2PO4 1.4mmol / L.
2) 0.01mol / L citrate buffer (CB, pH6.0, 1000ml): trisodium citrate 3g, citric acid 0.4g.
3) 0.5mol/L EDTA buffer (pH 8.0): dissolve 186.1g EDTA·2H2O in 700ml water, adjust to pH 8.0 with 10mmol/L NaOH, add water to 1000ml.
4) 1 mol/L TBS buffer (pH 8.0): Dissolve 121 g of Tris base in 800 ml of water, adjust to pH 8.0 with 1 N HCl, and add water to 1000 ml.
5) Enzyme digestion solution: a, 0.1% trypsin solution: prepared with 0.1% CaCl2 (pH 7.8). b. 0.4% pepsin solution: formulated with 0.1 N HCl.
6) 3% methanol-H2O2 solution: formulated with 30% H2O2 and 80% methanol solution.
7) Sealing agent:
a, glycerin and 0.5mmol / L carbonate buffer (pH 9.0 ~ 9.5) mixed in equal amounts;
b, oil and TBS (or PBS) preparation.
8) TBS/PBS pH 9.0~9.5, suitable for fluorescent microscope specimens; pH 7.0~7.4 is suitable for optical microscope specimens.
Immunohistochemistry experimental procedure:
1. Dewaxing and hydration
Prior to dewaxing, the tissue chip should be placed in a 60-minute or 60 ° C incubator for 20 minutes at room temperature.
1) The tissue chip is immersed in xylene for 10 minutes, and then replaced with xylene for 10 minutes;
2) Soaking in absolute ethanol for 5 minutes;
3) Soaking in 95% ethanol for 5 minutes;
4) Soaking in 70% ethanol for 5 minutes;
2, antigen repair
Paraffin-embedded tissue chip for formalin fixation.
1) Antigen heat repair
(1) High-pressure heat repair EDTA (pH 8.0) or 0.01 M sodium citrate buffer solution (pH 6.0) was added to boiling water. Cover the stainless steel pressure cooker, but do not lock it. Place the slide on the metal staining rack, slowly pressurize, soak the slide in the buffer for 5 minutes, then lock the lid and the small valve will rise. After 10 minutes, remove the heat source, place it in cool water, and open the lid when the small valve sinks. This method is suitable for antigen retrieval which is difficult to detect or nuclear antigen.
(2) Heat the boiling heat repair electric furnace or water bath to heat the 0.01M sodium citrate buffer solution (pH 6.0) to about 95 °C, and put it into the tissue chip and heat it for 10~15 minutes.
(3) Microwave heat repair In a microwave oven, a 0.01 M sodium citrate buffer solution (pH 6.0) is heated to boiling, and the tissue chip is placed, and the power is turned off, at intervals of 5 to 10 minutes, and repeated 1-2 times. Suitable antigens are: AR, Bax, Bcl-2, C-fos, X-jun, C-kit, C-myc, E-cadherin, Chromogranin A, Cyclin, ER, Heat shock protein, HPV, Ki-67, MDMZ, p53, p34, p16, p15, P-glycoprotein, PKC, PR, PCNA, ras, Rb, Topoismerase II and the like.
2) Enzymatic digestion methods commonly use 0.1% trypsin and 0.4% pepsin solution. Trypsin was preheated to 37 ° C before use, and the sections were preheated to 37 ° C, the digestion time was about 5 to 30 minutes; pepsin digestion was 37 ° C for 30 minutes. Applicable to immobilized antigens, including: Collagen, Complement, Cytokeratin, C-erB-2, GFAP, LCA, LN, etc.
The above specific steps are for reference only. If you want to know more about the immunohistochemistry experiment, you can click on for more details.
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