1. Fix sections with 5%glacial acetic acid/95%ethanol at 40C for 15 mins.
2. Wash sections with PBS (3 X lOmin.)
3. Treat sections with 0.2% bovine testicular hyaluronidase (Sigma type IV-S) in PBS for 30min atRT.
4. Wash sections with PBS (3 X 5min.)
5. Block the sections with 20% goat serum (Sigma) in PBS with 3% BSA.
6.Wash sections with PBS (3 X 5min.)
7. Apply 10 antibody, using 1:500 dilution (in PBS/3%BSA) and incubate at RT for
l-2hr. OR overnight at 40C.
8. Wash sections with PBS (3 X 5min.)
9. Apply FITC-conjugated goat anti-rabbit Ig (IgM+IgG,H+L) 20 antibody from Southern Biotechnology (diluted 1:80 in PBS/3%BSA with 5% normal mouse serum) and incubated for 1/2 hr at RT in the dark.
10. Wash sections with PBS (3 X 5min.) and mount in Vectashield(VECTOR).
Different Hyaluronidase concentrations have to be tested for the best result of different kinds of samples. You can do a titration from 0.l%-2%.
For 10 antibody, I tried the dilution from 1:50-1:1000, and found that the staining was still good at 1:500. May be you can do a titration too.
All steps were carried out in a moist chamber.