Sections were fixed in methanol at 4°C for 3 min, and then in acetone at 4°C for 3 min. All the following steps were performed at room temperature. The sections were airdried for 1 h and then rehydrated for 10 min in phosphate-buffered saline (PBS). All washes (3x10 min) between stages were performed in PBS. After the sections had been permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 5 min, potential non-specific binding sites were blocked with antibody dilution buffer [2% goat serum (Sigma-Aldrich) and 1% IgG-free bovine serum albumin (Sigma-Aldrich) in PBS] for 20 min at room temperature. Sections were then incubated with primary antibodies overnight at 4°C. After being washed, the sections were then incubated with secondary antibodies for 1 hour at room temperature. Following the final washing step, sections were mounted with Fluorescence Mounting Medium (Dako, North Sydney, Australia).
For immunostaining with two rabbit antibodies [anti-glial fibrillary acidic protein (GFAP) and anti-p75 neurotrophin receptor (p75NTR)] on the same section, a sequential immunostaining approach30 (link) was used [anti-GFAP+goat anti-rabbit IgG DyLight 488+ rabbit IgG (Sigma) + (mixture of anti-p75NTR +goat anti-rabbit IgG Alexa 555; incubated half hour before application)].
For immunostaining with two rabbit antibodies [anti-glial fibrillary acidic protein (GFAP) and anti-p75 neurotrophin receptor (p75NTR)] on the same section, a sequential immunostaining approach30 (link) was used [anti-GFAP+goat anti-rabbit IgG DyLight 488+ rabbit IgG (Sigma) + (mixture of anti-p75NTR +goat anti-rabbit IgG Alexa 555; incubated half hour before application)].
