Methods

Procedure for the immunolabelling of sections with Guinea pig antisera

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’s fluid or 10% formalin.
  • 0.1M Tris-buffered saline (TBS) pH 7.3.
  • SWant guinea-pig antiserum.
  • Biotinylated anti-guinea pig IgG.
  • Avidin-peroxidase (or streptavidin-fluorescence dye [e.g. Cy2 or Alexa 488])
  • Peroxidase substrate (e.g. Diaminobenzidine-HCl and H2O2 for immunoperoxidase.
  • Ethanol and Xylol (only for immunoperoxidase).
  • Mounting medium [Pertex, Histolab] for immunoperoxidase), [Hydromount, National Diagnostic] (for immunofluorescence).

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 0.1-0.2% Triton-X 100. Incubate for 1 to 3 days at 4°C (on a shaker for free-floating sections, in a humid chamber at 4°C for paraffin sections).
  2. Rinse in TBS 3 x 5 min.
  3. Apply biotinylated-anti guinea pig IgG (diluted according to the suggestions of the supplier) in TBS, with 10% carrier serum. Incubate at room temperature (RT) for 2 to 4 hours.
  4. Rinse in TBS 3 x 5 min.
  5. Apply the avidin-biotin-peroxidase complex (diluted according to the suggestions of the supplier) or Streptavidine-fluorophore (e.g. Cy2 or Alexa 488) in TBS with 10% carrier serum. Incubate for 1 to 2 hours at RT.
  6. Rinse in TBS 3 x 5 min.
  7. For immunoperoxidase: treat the section with a peroxidase substrate (e.g. Diaminobenzidine-HCl / H2O2).
  8. Rinse in TBS 3 x 5 min.
  9. Mount free floating sections on slides, eventually counterstain with cresyl-violet.
  10. For immunoperoxidase-staining dehydrate with ethanol and xylol and mount and coverslip in Entellan. For immunofluorescence-staining mount and coverslip in Hydromount.

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

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