Material :

• Free-floating cryostat or vibratome sections (fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4) or paraffin sections of tissue fixed with Bouin or 10% unbuffered formalin.
• 0.1M Tris-buffered saline (TBS) pH 7.3.
• SWant rabbit antiserum.
• Biotinylated anti-rabbit IgG.
• Avidin-peroxidase.
• Peroxidase substrate (e.g. Diaminobenzidine(HCl) and H2O2).
• Ethanol and Xylol.
• Mounting medium.

Method :

1. Apply the SWant antiserum diluted 1:1'000-1:5'000 (paraffin sections) or 1:5'000-1:10'000 (floating sections) in TBS with 10% carrier serum (e.g. calf or horse serum) and eventually 0.4% Triton-X 100 (particularly for vibratome sections). Incubate for 1 to 3 days at 4°C (on a shaker for free-floating sections, in a humid chamber for paraffin sections).
2. Rinse in TBS 3 x 10 min.
3. Apply anti-rabbit IgG-biotin (diluted according to the suggestions of the supplier) in TBS with 10% carrier serum. Incubate at room temperature (RT) for 1 to 4 hours.
4. Rinse in TBS 3 x 10 min.
5. Apply the avidin-biotin-peroxidase complex (diluted according to the suggestions of the supplier) in TBS with 10% carrier serum. Incubate for 1 to 2 hours at RT.
6. Rinse in TBS 3 x 10 min.
7. Treat the section with a peroxidase substrate (e.g. Diaminobenzidine HCl/ H2O2 ).
8. Rinse in TBS 3 x 10 min.
9. Mount free floating sections on slides, eventually counterstain with cresyl-violet.
10. Dehydrate with ethanol, xylol. Add mounting medium and coverslip.

Note : in case of excessive background-staining use higher dilutions of the SWant rabbit antiserum.