Immunohistochemical Analysis of Spinal Cord

Mice were anesthetized and perfused with 4% paraformaldehyde (PFA) in PBS. Spinal cords were removed, separated into portions as described above, and post-fixed in 4% PFA for additional 24 h. Tissues were cryo-protected in 20% glycerol for 24 h before 30-μm free-floating sections were prepared with a sliding microtome (Leica Microsystems, Wetzlar, Germany). Double immunolabeling was performed as previously described [35 (link)]. Briefly, sections were microwaved in 0.01 M citrate buffer (pH 6.0) followed by pretreatment with 1% Triton X in PBS. Sections were blocked with PBS containing 3% normal goat serum and 0.01% Triton X for 30 min and incubated with primary Ab diluted in blocking reagent overnight at 4°C (rabbit anti-GFAP, Dako, Carpenteria, CA, USA, 1:1,000; mouse anti-Iba-1, CCF Hybridoma Core, Cleveland, OH, USA, 1:250; goat anti-IL-6, R&D Systems, Minneapolis, MN, USA, 1:20). To confirm specificity, primary Abs were omitted on adjacent sections. The sections were washed and incubated with species-specific secondary Abs conjugated to FITC or Cy5 (Jackson ImmunoResearch, West Grove, PA, USA) for 2 h at RT. Sections were rinsed, mounted with VECTASHIELD (Vector Labs, Burlingame, CA, USA) and examined on a Leica TCS confocal microscope (Leica Microsystems). Images were analyzed offline with Volocity software version 6.1.2 (PerkinElmer, Waltham, MA, USA).