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Immunohistochemistry of Pax7 antibody
- Deparaffinize the sections and rehydrate to distilled water
- Unmask the antigen using unmasking solution (VECTOR LABORATORIES, Catalog Number H-3300), according to the proportion 15ml of the stock Antigen Unmasking Solution to 1600ml of distilled water, Firstly boiled the the apropriate volume of distiledwater and then added proportional unmasking solution, kept the the temperature around 95° C, did unmasking for 20 minutes, and then transfered the slides to distilled water for 2 times followed into PBS (0.01MPB containing 0.3M NaCl, PH7.4)
- Blocking the nonspecific reaction, the sections were incubated in the 10% normal horse serum diluted with PBST (the above PBS added TritonX-100 to final concentration 0.3%) for 30 minutes to 60 minutes.
- Wipe off the blocking solution, incubate with PAX7 mouse monoclonal antibody in a titre1:100 diluted with PBST containing 1% normal horse serum overnight at 4° C. The primary antibody developed by Dr. A. Kawakami was obtained from the Developmental Studies Hybridoma Bank developed under Department of Biological Sciences, Iowa City, IA 52242.
- The slides were washed with flow PBS
- Incubated the sections with biotinylated horse antimouse antibody (Vector Laboratories, Vectastain Elite, ABC Kit PK6102) in a titre 1:200 diluted with PBST at room temperature for 2 hours.
- The slides were washed with flow PBS.
- Incubated the sections with ABC reagent (Vector Laboratories, Vectastain Elite, ABC Kit, PK6102) in a titre 1:100 diluted with PBST at room temperature for 2 hours. The ABC reagent was prepared 30 minutes before using.
- The slides were washed with flow PBS.
- The slides were washed with 0.1M Tris-HCl, 0.15M NaCl, PH7.6.
- The substrate DAB (3,3'-Diaminobenzidine)( Sigma, FAST DAB Cat. D4293) prepared, performed the color reactin for 10 minutes.
- Wash the slides, dehydrated, transparented and covered with Permount mounting medium.
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