Rabbit anti gfap antibody

Neurons were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS) plus 120 mM sucrose for 20 min at 37°C. Neurons were incubated in 20 mM glycine for 5 min, rinsed and permeabilized with 0.2% Triton X-100 for 5 min at room temperature, and then blocked for 30 min with 2% bovine serum albumin (BSA). Chicken anti-total cofilin/ADF antibody AE774 1∶300 (kind gift from J.S. Condeelis), rabbit anti-phosphorylated cofilin/ADF 1∶300 (Abcam, Cambridge, MA; catalog # ab12866), mouse anti-total cofilin/ADF antibody 1∶100 (Abcam, Cambridge, MA; catalog # ab54532), chicken anti-MAP2 antibody 1∶1000 (Lifespan Biosciences), rabbit anti-GFAP antibody 1∶500 (Dako, Denmark), rabbit anti-MAP2 antibody 1∶2000 (S. Halpain), rat anti-HA antibody 1∶500 (Roche Applied Science), rabbit anti-LIMK antibody 5079 1∶500 (kind gift from G.N. Gills), used to detect overexpressed wt-LIMK. All antibodies were incubated for 1 hr at room temperature, and, following rinsing with PBS, were incubated with AlexaFluor-conjugated secondary antibodies (Invitrogen, Molecular Probes) for 45 min at 37°C. To label F-actin, AlexaFluor488-, 568- or 647-phalloidin at 1∶1000 (Invitrogen, Molecular Probes) was incubated for 2 hr at room temperature in the presence of 2% BSA. Finally the coverslips were washed twice with PBS and mounted using Aqua-Mount (Thermo Scientific).

Coronal sections (4 µm thick) were cut and deparaffinized using routine protocols before being incubated with a monoclonal anti-Iba-1 antibody (1:500 dilution; Thermo Fisher Scientific, Waltham, Massachusetts (MA), USA) or a rabbit anti-GFAP antibody (1:1000 dilution; Dakocytomation, Glostrup, Denmark). Primary antibody binding was detected with biotinylated horse anti-mouse or goat anti-rabbit IgG (Vector Elite kit; Burlingame, CA, USA), respectively. Immunoreactivity was measured using an avidin–biotin peroxidase complex (Vector Elite kit). The peroxidase reaction was developed using the diaminobenzidine substrate assay (Vector Elite kit). The slides were counterstained with Harris’ hematoxylin, before being mounted with Canada balsam (Sigma-Aldrich) [17 (link)].

Eyes were enucleated and fixed by immersion in 4% paraformaldehyde for 1 hour. Retinas were isolated by dissection, and flat mounted following incubation with biotinylated isolectin B4 (Sigma-Aldrich, 1/200 from 1mg/ml stock) and Alexa Fluor 546 conjugated streptavidin (Life Technologies). Images were taken by using a 10x objective and tilescan options. The extent of retinal vascular regression (central avascular area) and pre-retinal vascularization (NV) were measured using Image J [5 (link)], by quantification of manually selected area and intensity threshold- selected particles, respectively. For GFAP staining, eyes were fixed in 1% paraformaldehyde for 1 hour, embedded in OCT (RA Lamb) and frozen in isopentane precooled in liquid nitrogen. Sections of 18 μm of thickness were incubated with rabbit anti-GFAP antibody (Dako) and anti-rabbit-488 secondary antibody (Life Technologies). Lungs of the nursing mothers were dissected and fixed by immersion in 4% paraformaldehyde for 12 hours. DeadEnd^™ Fluorometric TUNEL System kit (Promega) was used to determine apoptosis in 10, non-consecutive lung cryosections. Alternatively, cryosections were incubated with anti-rat CD45 (Millipore) or Van Gieson stain.

MK-801 and 2,3,5-triphenyltetrazolium chloride (TTC) were purchased from Sigma (St. Louis, MO, USA). Maslinic acid (96.5 percent purity, No. 0060324) was supplied by Center for Drug Discovery, China Pharmaceutical University. Rabbit anti-GFAP antibody was obtained from DAKO (Glostrup, Denmark), and rabbit anti-GLAST and anti-GLT-1 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).