Procedure for the immunolabelling of sections with mouse monoclonal antibodies
Material
- Free-floating cryostate or vibratome sections (40-80 µm) of fixed tissue* (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4) or paraffin sections (3-5 µm) of tissue fixed with 10% unbuffered formalin.
- 0.1M Tris-buffered saline (TBS) pH 7.3.
- SWant monoclonal mouse antiserum.
- Biotinylated anti-mouse IgG.
- Streptavidin-peroxidase or Streptavidin conjugated to CY2, Cy3, Cy5, or other fluorescent dyes.
- Peroxidase substrate (e.g. Diaminobenzidine(HCl) and H2O2).
- Ethanol and Xylol.
- Mounting medium (e.g. Eukitt for immunhistochemistry, Hydromount for immunofluorescence).
Method
- Apply the SWant mouse-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 0.2% 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).
- Rinse in TBS 3 x 5 min.
- Apply biotinylated anti-mouse 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.
- Rinse in TBS 3 x 5 min.
- Apply the streptavidine-biotin-peroxidase complex (diluted according to the suggestions of the supplier) or the Streptavidine-fluorescent complex in TBS with 10% carrier serum. Incubate for 1 to 2 hours at RT.
- Rinse in TBS 3 x 5 min.
- The immunofluorescent labelling is terminated at this point and the section can be mounted on slides and coverslipped with an aqueous medium (e.g. Hydromount).
- The immunohistochemical reaction is concluded by treating the section with a peroxidase substrate (e.g. Diaminobenzidine HCl / H2O2 ) under continuous microscopic observation.
- Rinse in TBS 3 x 5 min.
- Mount free floating sections on slides, eventually counterstain with cresyl-violet.
- Dehydrate immunohistochemically treated sections with ethanol and xylol. Add mounting medium (Eukitt) and coverslip.
Note : in case of excessive background-staining, use higher dilutions of the SWant mouse antiserum.
* Without prior fixation the highly soluble Ca2+-binding proteins are immediately lost during sectioning.