Rabbit anti mouse gfap

Eyes from animals treated with various test compound or solvent control were embedded in Optimal Cutting Temperature (OCT) compound at −80°C. Eyes were sectioned (9 µm) using a cryostat, sections were placed on glass slides, and allowed to dry for 2 h. For immunostaining, sections were fixed in cold acetone (4°C) on ice for 10 min, followed by three washes in PBS, 5 minutes each. Slides were incubated in blocker solution (1% BSA, 0.2% skim milk, and 0.3% Triton X-100 in PBS) for 15 minutes at room temperature, and then incubated with rabbit anti-mouse-GFAP (Cat#: Z0334; DAKO, Denmark; 1:500 dilution) overnight at 4°C in humid environment. After three washes in PBS, 5 min each, samples were incubated with appropriate secondary antibodies for 2 h at room temperature (Jackson Immunoresearch, West Grove, PA; 1:500 dilution). As a control, some sections were incubated only with secondary antibody. Sections were washed three times in PBS, covered with PBS: glycerol (2 vol/1 vol), and mounted with a coverslip. Retina sections were examined using a Zeiss fluorescence microscope and images were captured in digital format.

Spinal cord sections mounted on slides for immunofluorescence were rinsed in PBS for 5 min to remove residual OCT. After washing, samples were blocked in serum of the host of the secondary antibody (5% serum and 0.3% BSA in PBS with 0.2% Triton-X 100) and then incubated overnight with rabbit anti-mouse Iba1 (1:500, Wako), mouse anti-mouse iNOS (1:500, BD Biosciences), mouse anti-mouse Arg-1 (1:500, BD Biosciences), rat anti-mouse CD86 (1:500, BD Biosciences), goat anti-mouse CD206 (1:100, R&D Systems), rabbit anti-mouse GFAP (1:1000, Dako) rabbit anti-mouse NG2 (1:500, a generous gift from the Levine lab), mouse anti-mouse CC1 (1:100, EMD Millipore), rat anti-mouse MBP (1:50, Bio-Rad), or rabbit anti-mouse neurofilament-L (1:100, EMD Millipore) in 0.3% BSA in PBS with 0.2% Triton-X 100. After washing with PBS, sections were incubated with fluorescence-conjugated FITC or Cy3 goat anti-rabbit, goat anti-mouse, goat anti-rat, or donkey anti-goat antibody for 1 h at room temperature, washed three times with PBS, and mounted using Fluoromount-G with DAPI (Southern Biotech, USA). The sections were imaged using a Nikon Eclipse E600 microscope and Zeiss LSM 510 confocal microscope.