Standard protocol for immunocytochemistry of PFA fixed cultured neurons
- Phosphate buffer saline (PBS), pH 7.4
- 4 % Paraformaldehyd in PBS, pH 7.4 / 4 % sucrose
- Blocking solution: 10 % normal serum / 0.1 % Triton X-100 / PBS (normal serum from the host-species of the secondary antibodies is recommended for blocking).
- Incubation solution: 3 % normal serum / 0.1 % Triton X-100/ PBS (Triton may be omitted; normal serum from the host-species of the secondary antibodies is recommended for blocking).
- Mounting medium
- Wash coverslips twice with PBS.
- Fix cells with 3 ml/well paraformaldehyde solution for 15-20 min at RT.
- Wash three times with PBS for 10 min.
- Incubate for 30 min with blocking solution.
- Put a piece of parafilm on wet Whatman paper and apply 200 µl of primary antibody solution in incubation solution (appropriate dilution must be determined experimentally).
- Put coverslips upside down on antibody-solution and incubate for 2 h at RT.
- Transfer slips to multi-well plate and wash three times with PBS for 10 min.
- Repeat steps 5-7 with secondary antibody (optimal dilution must be determined experimentally). Avoid bright light when working with the secondary antibody to minimize photo bleeching of the fluorescent dye.
- Mount coverslips on slides and microscope