Invitrogen prolong gold antifade mountant with dapi

Spinal cord tissue segments (S3, injury section) from the injury site were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) overnight(Haque et al., 2017a (link)). Tissue was then embedded in paraffin, sectioned, and mounted onto slides for analysis. Following epitope retrieval, non-specific binding sites were blocked with the serum of the secondary antibody host for 1h at room temperature (Samantaray et al., 2015 (link)). Tissues were incubated with vimentin (1:500, Abcam ab92547), Iba1 (1:250, Abcam ab5076), GFAP (1:250, Santa Cruz sc-9065), Cox-2 (1:100, Abcam ab15191), caspase-1 (1:200, Abcam ab74279), CSPG (1:200, Sigma C8035), or NFP (1:1000, Sigma Aldrich N4142) antibodies overnight at room temperature. Sections were then incubated with Texas Red® Goat Anti-Rabbit IgG Antibody (Vector Laboratories, Inc., Burlingame, CA), DyLight® 488 Anti-Goat IgG (H+L) (Vector Laboratories, Inc., Burlingame, CA), Texas Red® Anti-Mouse IgM Antibody (Vector Laboratories, Inc., Burlingame, CA), or Fluorescein Anti-Rabbit IgG (Vector Laboratories, Inc., Burlingame, CA) for 1h in the dark. Slides were mounted with 1 drop of Invitrogen™ ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific) and coverslipped. After staining, SCI tissues were viewed under a fluorescence microscope with representative images taken at 20× magnification.

M1 cells were cultured on poly-d‐lysine‐coated (Sigma-Aldrich) coverslips, fixed with 4% paraformaldehyde (Sigma‐Aldrich) in PBS (pH 7.4) for 10 min, washed three times with PBS, and incubated with blocking buffer containing 10% normal goat serum (Life Technologies) in PBS and 0.1% Triton X‐100 for 30 min at room temperature. After removing the blocking buffer, cells were incubated overnight with primary antibodies against vimentin (1:500, Abcam ab92547) and GFAP (1:200, mouse monoclonal; Sigma‐Aldrich‐Aldrich Cat# G3893) at 4 °C. After washing three times with PBS at room temperature, cells were incubated with fluorophore‐conjugated secondary antibodies (Sigma-Aldrich) that recognized primary antibodies. DAPI (Thermo Fisher Scientific) was used to label cell nuclei. Slides were mounted with 1 drop of Invitrogen^™ ProLong^™ Gold Anti-fade Mountant with DAPI (Thermo Fisher Scientific) and cover slipped. After staining, cells were viewed under a fluorescence microscope with representative images taken at × 20 magnification.