Goat anti gfap antibody

After injecting an overdose of urethane, rat L4–L5 spinal segments were removed immediately, cut into ipsilateral and contralateral quadrants, frozen in liquid nitrogen and stored at −80 °C for further analysis. The samples were homogenized using an ultrasonic cell processor in SDS sample buffer containing proteinase inhibitors and PMSF. Equal amounts of the protein sample were separated by performing 10% tris–tricine SDS–PAGE and were transferred onto PVDF membranes (Millipore). The membranes were blocked with 5% nonfat milk for 2 h at RT. The membranes were incubated overnight at 4 °C with rabbit anti-IFN-γ antibody (1:500 dilution; Abcam), JNK (1:1000 dilution; Abcam), pJNK (1:1000 dilution; Abcam) or goat anti-GFAP antibody (1:10000 dilution; Sigma) and then with HRP-conjugated secondary antibodies (1:1000 dilution; Pierce) for 2 h at RT. Bands were developed using enhanced chemiluminescence (Pierce) and were visualized using ChemiDoc XRS system (Bio-Rad). All western blotting were performed at least 3 times, and similar results were obtained from all the analyses. The density of band area was quantified using Image Lab 3.0. A same-sized square was drawn around each band to measure its density, and the background of that band was subtracted. GAPDH expression was used as a loading control for protein expression.

IHC and IF were performed as previously described (31 (link)). In brief, mice were deeply anesthetized and transcardially perfused with PBS and 4% paraformaldehyde (PFA). Brains were excised, postfixed in 4% PFA overnight, saturated in 30% sucrose solution, and sectioned with a cryotome at 30 μm. IHC staining of microglia was performed using anti-IBA1 antibody (Wako Chemicals, Neuss, Germany) and imaged using a Zeiss AXIO Scope A1 equipped with a Plan-APOCHROMAT 20×/0.8 objective. Images were acquired at room temperature with the AxioRel Vision 4.8 software and quantified in ImageJ. For double IF goat anti-GFAP antibody (Sigma-Aldrich) and rabbit anti-MGL antibody (kind gift from Ken Mackie, Indiana University Bloomington) and anti-goat (Alexa Fluor 488-conjugated) and anti-rabbit (Alexa Fluor 594-conjugated) secondary antibodies were used. Sections were imaged by confocal laser scanning microscopy using a Leica TCS SP5 II equipped with a HCX PL APO 63 × 1.3 NA objective. Images were obtained at room temperature with Leica LAS AF software.