Immunohistochemistry protocol

(1) Instruments and equipment 1) 18cm stainless steel pressure cooker or electric furnace or medical microwave oven;
2) Water bath

(B), reagent 1) PBS buffer (pH 7.2 ~ 7.4): NaCl 137mmol / L, KCl 2.7mmol / L, Na2HPO4 4.3mmol / L, KH2PO4 1.4mmol / L.
2) 0.01 mol/L citrate buffer (CB, pH 6.0, 1000 ml): trisodium citrate 3 g, citric acid 0.4 g.
3) 0.5 mol/L EDTA buffer (pH 8.0): 186.1 g of EDTA·2H2O was dissolved in 700 ml of water, adjusted to pH 8.0 with 10 mmol/L NaOH, and water was added to 1000 ml.
4) 1 mol/L TBS buffer (pH 8.0): 121 g of Tris base was dissolved in 800 ml of water, adjusted to pH 8.0 with 1 N HCl, and water was added 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 fluorescence microscopy specimens; pH 7.0-7.4 is suitable for optical microscope specimens.

(C), the operation process 1, dewaxing and hydration before dewaxing, the tissue chip should be placed in the oven for 60 minutes or 60 ° C incubator for 20 minutes.
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 Add EDTA (pH 8.0) or 0.01 M sodium citrate buffer solution (pH 6.0) 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.
3, immunohistochemical staining SP method 1) dewaxing, hydration;
2) Wash PBS 2 to 3 times for 5 minutes each;
3) 3% H2O2 (80% methanol) was added dropwise to TMA and allowed to stand at room temperature for 10 minutes;
4) Wash PBS 2 to 3 times for 5 minutes each;
5) antigen retrieval;
6) Wash PBS 2 to 3 times for 5 minutes each;
7) Add normal goat serum blocking solution to room temperature for 20 minutes. Remove excess liquid.
8) 50 μl of I anti-drop was added dropwise, and allowed to stand at room temperature for 1 hour or 4 ° C overnight or 37 ° C for 1 hour.
9) After overnight at 4 ° C, it was rewarmed at 37 ° C for 45 minutes.
10) Wash PBS 3 times for 5 minutes each;
11) Adding II anti-40-50 μl dropwise, standing at room temperature, or 37 ° C for 1 hour;
12) 0.05% tween-20 can be added to the II antibody.
13) Wash PBS 3 times for 5 minutes each;
14) DAB color development for 5 to 10 minutes, master the degree of dyeing under the microscope;
15) Rinse with PBS or tap water for 10 minutes;
16) Hematoxylin counterstaining for 2 minutes, hydrochloric acid alcohol differentiation;
17) Rinse with tap water for 10 to 15 minutes;
18) Dehydration, transparency, sealing, and microscopic examination.
SABC method 1) Dewaxing and hydration.
2) Wash the PBS twice for 5 minutes each.
3) Dissolve fresh 3% H2O2 in distilled water or PBS, block at room temperature for 5 to 10 minutes, and wash 3 times with distilled water.
4) Antigen retrieval.
5) Wash in PBS for 5 minutes.
6) Add normal goat serum blocking solution at room temperature for 20 minutes. Remove excess liquid.
7) I anti-drug was added dropwise at room temperature for 1 hour or overnight at 4 ° C or 1 hour at 37 ° C (over the night at 4 ° C for 45 minutes at 37 ° C).
8) Wash PBS three times for 2 minutes each time.
9) Add biotinylated secondary antibody dropwise at 20 ° C to 37 ° C for 20 minutes.
10) Wash the PBC 3 times for 2 minutes each time.
11) Add the reagent SABC dropwise at 20 ° C to 37 ° C for 20 minutes.
12) Wash PBS 4 times for 5 minutes each time.
13) DAB color development: DAB color development kit or self-matching color developer (the degree of color development under the microscope).
14) Washed with distilled water. Hematoxylin was counterstained for 2 minutes, and hydrochloric acid alcohol was differentiated.
15) Dehydration, transparency, sealing, and microscopic examination.