Immunohistochemistry for Microglia Assessment

After BI, GTS-21 or saline-treatment (n = 3), IHC to detect microglia changes was performed using anti-Iba1. ^{20 (link),30 (link)} Rats were anesthetized as described, chest opened, a needle was inserted through the heart and ascending aorta perfused via infusion pump with 200ml of 0.01m phosphate buffered saline (PBS, pH 7.35) followed by 300ml paraformaldehyde in 0.1 M in PBS. Then, spinal segments were excised, post fixed (4% formaldehyde) and kept in sucrose (10%) for 4h, sucrose(20%) for 8h and then sucrose(30%) overnight. After tissue dehydration, spinal segments were paraffin embedded, sectioned with a cryostat (8μm thick), mounted on Shandon™ Polysine slides (Fisher-Scientific, Waltham, MA) and stored at −20^oC. All IHC sections were treated under the same conditions on the same day to minimize the between-group variability. Sections were blocked (1.5% goat serum and 0.04% Saponin in 1% bovine serum albumin) for 1h, incubated overnight at 4^oC with primary antibody, rabbit anti Iba1(1:150, Wako-Pure Industries, Richmond, VA) and incubated for 90min with anti-rabbit Alexa 568 (1:300; Invitrogen, Grand Island). Randomly-selected IHC images were scanned using a confocal microscope (Zeiss LSM 800; Carl Zeiss Microscopy, Thornwood, NY). Photoshop program (Volocity 6.3, Perkin Elmer, Inc) was used for quantitation of expression of Iba1.