Immunocytochemistry of Schwann Cells and Fibroblasts

Schwann cells or fibroblasts were seeded on 8-well chamber slides (Ibidi) coated with poly-L-lysine and laminin, as described.⁹ (link) Cells were harvested 24 to 48 h later and fixed in 4% paraformaldehyde (EM Sciences) for 15 min at RT. Fixed cells were permeabilized in PBS containing 1% bovine serum albumin and 0.2% Triton X-100 for 30 min at RT, and further incubated with anti-p75 NGFR overnight at 4°C in the dark. The next day, cells were washed in PBS containing 1% BSA two times for 5 min at RT, incubated with anti-phalloidin for 1 h in the dark at RT, and washed again as described above. Cells were mounted with ProLong Gold Antifade Mountant with DAPI (Thermo) and coverslipped using Gold Seal Cover Glass (EM Sciences). Images were acquired using a Cytation 5 cell imaging reader (BioTek).