Immunohistochemistry of Pax3 antibody with ABC
(Fuzi Jin and Jon Epstein)
- Deparaffinize the sections through xylene 10' X 3, and gradually rehydrate by passing through ethanol 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, First boil the apropriate volume of distiled water and then added proportional unmasking solution, keep the temperature around 95° C, unmask for 20 minutes, and then transfer the slides to distilled water 2 times followed by buffer A (0.3M NaCl, 0.01M sodium phosphate, PH7.4)
- To block nonspecific binding, the sections are incubated in 10% normal goat serum diluted with buffer B (the above buffer A with TritonX-100 to final concentration 0.3%) for 30 to 60 minutes.
- Wipe off the blocking solution, incubate with PAX3 rabbit polyclonal antibody at a titre of 1:3000 diluted with buffer B containing 1% normal goat serum overnight at 4° C.
- Wash with buffer A
- Incubated the sections with biotinylated goat antirabbit antibody (Vector Laboratories, Vectastain Elite, ABC Kit PK6101) at a titre of 1:200 diluted with buffer B at room temperature for 2 hours.
- Wash with buffer A.
- Incubated the sections with ABC reagent (Vector Laboratories, Vectastain Elite, ABC Kit, PK6101) in a titre 1:100 diluted with buffer B at room temperature for 2 hours. The ABC reagent is prepared 30 minutes before using.
- Wash with buffer A..
- Wash with 0.1M Tris-HCl, 0.15M NaCl, PH7.6.
- Perform DAB color reaction for 10minutes ( Sigma, FAST DAB Cat. D4293).
- Wash the slides, dehydrate, pass through xylene and cover slip with Permount mounting medium.
Immunohistochemistry of Pax3 antibody with fluorescence
For immunofluorescence, all the proceedures are similar to the ABC method, but use Pax 3 antibody at 1:800 dilution. The second immunofluorescence antibody was bought from Jackson ImmunoResearch Laboratories Inc. FITC labeled (Cat:111-095-144), or Texas Red labled (Cat: 111-075-144). The titre, was 1:200.