Protocol for immunohistochemistry - cryo sections
- 0. 9% saline
- Fixation solution: 4 % paraformaldehyde / 50 mM phosphate buffer, pH 7.2 / 150 mM NaCl.
- 0.1 M Tris-HCl, pH 7.2
- 20 % sucrose in 0.1 M Tris-HCl, pH 7.2
- TBS: 20 mM Tris, pH 7.2 / 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
- Transcardially perfuse with 0.9 % saline for 1 min.
- Perfuse with fixation solution for 10-20 min with a rate of 12 ml/min.
- Fix tissue in fixation solution overnight.
- Rinse tissue in 0.1 M Tris-HCl, pH 7.2 and change buffer three times.
- Incubate tissue for 12-18 h in 20 % sucrose, 0.1 M Tris-HCl, pH 7.2.
- Freeze tissue with TISSUE-TEK using a cryostat.
- Cut tissue (6-10 µ) and mount sections on Super Frost Ultra Plus® slides.
- Allow frozen sections to dry for 15 min at RT.
- Block for 1-2 h in blocking solution.
- Incubate with primary antibody diluted in incubation buffer overnight at 4°C (optimal dilution must be determined experimentally).
- Wash three times for 10 min in TBS.
- Incubate with secondary antibody diluted in incubation buffer for 1 h at RT. Avoid bright light when working with the secondary antibody to minimize photo bleaching of the fluorescent dye.
- Wash three times in TBS for 10 min.
- Mount with mounting medium and microscope.