Immunohistochemistry experimental steps - Database & Sql Blog Articles

1. Reagent preparation 1. 0.01 M PBS (pH 7.34) 9.0 g NaCl + 50 ml 0.2 M PB plus double distilled water to 1000 ml; 1000 ml 0.2 M PB (pH 7.4) = 5.93 g NaH2PO4 · 2H2O + 58.02 g Na2HPO4· 12H2O in 1000 ml double distilled water or =190 ml A + 810 ml B (A. 0.2 M NaH2PO4·2H2O: 15.6 g in 500 ml ddH2O; B. 0.2 M Na2HPO4·12H2O: 71.632 g in 1000 ml dH2O). 2. Citrate Buffered Saline (0.01 M citrate buffer, pH 6.0) 28 ml A + 72 ml B + 200 ml ddH2O (A. Citrate acid (citric acid): 10.5 g plus double distilled water to 1000 ml; B. Citrate sodium: 29.41 g plus double distilled water to 1000 ml). 3. The cell permeable solution was prepared by mixing a final concentration of 0.3% hydrogen peroxide and 0.3% Triton X-100. The preparation method is to first heat the microwave in 36 ml PBS, then add 120 ul TritonX-100, and heat for a while, then cool to 0.4 ml 30% H2O2 before use. 4. 5% sheep serum or blocking serum, diluted with PBS . 5. 0.05% DAB (with light) containing 0.03% H2O2: using 20 x DAB (1%, 10 mg/ml) 5 ul + 0.1 ul 30% H2O2 + 95 ul PBS. 6. Primary and secondary antibodies were diluted with PBS. 7. Xylene, gradient Ethanol (100% × 2, 95%, 80%, 70%, 50%), double distilled water, neutral gum (sealing agent). 8. Hematite dyeing solution II. Operation steps 1. Dewaxing and hydration (1) 60 °C × 20 min → Xylene 2 × 10 minutes; (2) 100% absolute ethanol: 2 × 5 minutes; (3) 95 % ethanol 2 minutes; (4) 80% ethanol 2 minutes; (5) 70% ethanol 2 minutes; (6) distilled water: 5 min; (7) PBS washed 3 times x 3 min. 2, cells are permeable, blocking endogenous peroxidase (1) infiltrated with closed permeable solution for 30 min (RT protected from light). The method was to preheat with 40 ml of PBS and 120 ul of Triton X-100 for a few minutes, then add 400 ul of 30% H2O2 before use. (2) Wash the PBS solution 3 times × 3 min. 3, antigen retrieval exposure antigen determinant (1) slice into 0.01 M sodium citrate buffer solution (pH 6.0), after heating in a microwave oven for 4 min to boiling, and then heating for about 6 min × 4 times, each time Fill the liquid at intervals to prevent dry film. 4. Block the non-specific protein (1) Wash the PBS solution 3 times × 3 min; (2) Take out the slice, blot the surrounding water with a filter paper, draw a circle around the tissue with a tissue pen, and drop the tissue into the circle. % sheep serum (consistent with the source of secondary antibody), put into a wet box, room temperature 30 (10 ~ 30) min; 5, primary antibody incubation (1) remove the sheep serum on the slice, dry the residual serum around the tissue with filter paper Immediately after adding the diluted primary antibody (1:250, 500, 1000), put it into the wet box for 1 hour at room temperature, then 4 degrees overnight, and take it out from the refrigerator for 45 minutes at 37 °C. 6. Incubation of secondary antibody (1) Pour the primary antibody and wash it with PBS for 5 min×5 times; (2) Use water filter paper to absorb the water around the circle, add the diluted secondary antibody, and put it into a 37 °C constant temperature oven. Min (recycling). (3) Wash 5 times with PBS × 5 min; 7. SP reaction (1) Add SP and put in 37 degree oven for 30 min. (2) Wash 5 times with PBS × 5 min; 8. Color (1) Add DAB (rapid drop, observe the staining, pour out the dye solution), and control the coloring time to about 5 min (3 to 10 min) The color control time is observed by the microscope. 0.05% DAB (protected from light) (1:20 stock solution): 5 ul 20 x DAB + 0.1 ul 30% H2O2 + 95 ul PBS. Preparation of 1% DAB stock solution: 50 mg DAB + 5 ml 0.05 mol/L PBS was fully dissolved and stored at -20 °C. 9, counterstaining, dehydration, transparent, sealing (1) with PBS 3 times × 3min, washed with double distilled water for 5 min; (2) add a large drop of hematoxylin dye solution, nucleoprotein staining for a few seconds, cytoplasm or The membrane protein was stained with tap water for 20 s, washed with double distilled water for 5 min, and then returned to the blue for 5 min with PBS. (3) Dehydration (4) Transparent Xylene 1×(1-2) min→Xylene 2×(1-2) min(5) Sealed neutral gum.