Protocol for immunohistochemistry - paraffin sections
- 10 mM citrate buffer: pH 2.5-6.0 (varies from antibody to antibody)
- TBS: 20 mM Tris, pH 7.3 / 150 mM NaCl
- Blocking solution: 10 % normal serum / 0.3 % Triton-X 100 in TBS (normal serum from the host-species of the secondary antibodies is recommended for blocking).
- Incubation buffer: 5 % normal serum / 0.3 % Triton-X 100 in TBS (normal serum from the host-species of the secondary antibodies is recommended for blocking).
- Mounting medium
Deparaffinization and rehydration:
- Incubate sections with xylene twice for 5 min.
- Rehydrate sections through a series of ethanol concentration ranging from 100 % to 70 % (10 % steps) with a minimum of 10 min/concentration.
- Rinse sections in TBS.
- Water bath sections in 10 mM citrate buffer at 95°C for 40 min to retrieve antigens that are masked through the embedding process.
- Cool slides slowly to RT.
- Rinse slides in TBS.
- Block in blocking solution for 30 min at RT.
- Remove blocking solution and incubate sections overnight at 4°C with the primary antibody diluted in blocking solution (optimal dilution must be determined experimentally).
- Remove primary antibody and wash thoroughly with TBS three times for 10 min.
- Incubate with the secondary antibody diluted in incubation solution for 1 h at RT. Avoid bright light when working with the secondary antibody to minimize photo bleaching of the fluorescent dye.
- Remove secondary antibody and wash thoroughly with TBS three times for 10 min.
- Mount slices mounting medium and microscope.