Protocol for immunohistochemistry - low pH fixative
- 0. 9% saline
- Fixation solution: 1-2 % paraformaldehyde / 100 mM Na acetat buffer, pH 6.0.
- Phosphate buffer: 100 mM phosphate buffer, pH 7.3
- TBS: 20 mM Tris, pH 7.3 / 150 mM NaCl
- Blocking solution: 10 % normal serum in TBS (normal serum from the host-species of the secondary antibodies is recommended for blocking).
- Incubation buffer: 2 % normal serum / 0.1 % 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.
- Cut 60 µm thick coronal sections with a vibratome.
- Wash in phosphate buffer for 10 min.
- Wash in TBS for 10 min
- Block for 1 h in blocking solution.
- Incubate with primary antibody diluted in incubation buffer overnight at RT (optimal dilution must be determined experimentally).
- Wash three times for 10 min in TBS .
- Incubate with secondary antibody diluted in incubation buffer for 2 h at RT. Avoid bright light when working with the secondary antibody to minimize photo bleaching of the fluorescent dye.
- Wash three times for 10 min in TBS.
- Mount with mounting medium and microscope.