Immunohistochemical operation procedure (PAP method, ABC method) 1. Dewaxing to water: xylene (1), (2) dewaxing 15min each â†’ absolute ethanol (1), (2) 5min each â†’ 90% ethanol 5min â†’ Water washing â†’ PBS5min2. Antigen repair: 1) Microwave repair: microwave medium and high-grade 5min 3 times â†’ natural cooling to room temperature â†’ PBS once again 3 times? Min, 2) enzyme digestion: 0.1% trypsin incubation at 37 â„ƒ for 30min â†’ PBS5min 3. Block endogenous peroxidase: set 3% 3 times? Soak in hydrogen peroxide for 15min â†’ PBS5min 4. Block: 10% normal secondary antibody serum at room temperature for 10min 5. Primary antibody incubation: remove the serum and add primary antibody, place in a wet box Overnight at 4 Â° C â†’ 3 times for PBS5min? 3 times? 6. Secondary antibody incubation: add secondary antibody after wiping, incubate in a humidity box at 37 Â° C for 30min â†’ PBS5min III. Immunohistochemical operation: 1.4% paraformaldehyde is routinely perfused and fixed, and the material is taken and placed in 20% sucrose solution (4 Â° C) overnight. Wax block production. Slice, patch. After washing with 0.01MKPBS for 5min Ã— 3; 2. Add the prepared 0.3% methanol solution of hydrogen peroxide (methanol 80ml + 0.01MKPBS 100ml + 30% hydrogen peroxide) for 30min to eliminate the effect of endogenous peroxidase, wash with 0.01MPBS 5min Ã— 3; 3. Add the prepared 0.3% Triton X100 (30% Triton X100 + 0.01MKPBS 100ml) for 30min to increase the permeability of the cells, wash with 0.01MKPBS for 5min Ã— 3; 4. Add the serum dilution (bovine serum) Albumin (1.00g + 0.01MPBS 100ml + sodium azide 0.08g) diluted primary antibody, stored at 4 â„ƒ for 24-48h; absorb the antibody, washed with 0.01MKPBS for 5min Ã— 3; 5. Add a secondary antibody diluted with 0.01MKPBS, incubate at room temperature for 2h . Wash with 0.01MKPBS for 5min Ã— 3; 6. Add antibodies such as ABC complex and incubate at room temperature for 2h, wash with 0.01MKPBS for 5min Ã— 3; quickly rinse three times with distilled water; 7. Add color developing solution for immunohistochemical color development, the time is normal No more than 30min, can be observed under the microscope from time to time, when the cells are colored and the background color is lighter, immediately absorb the coloring solution, rinse three times with distilled water and then add 0.01MKPBS to stop the reaction; 8. After gradient alcohol dehydration, mount the film , Take pictures. Source: Bio Show Forum
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