Rabbit anti hif 1α polyclonal antibody

Fixed frozen sections of spinal cord from mSOD1^G93A mice (10 μm thick) were prepared as previously described^{32 (link)}. Hypoxia-induced factor 1-alpha (HIF-1α) expression was evaluated in paraffin-embedded 6-μm–thick sections of the lumbar cord from ALS patients and 10 μm–thick fixed frozen sections of spinal cord of mSOD1^G93A mice as previously described^{41 (link)}. The following primary antibodies were used: rabbit anti–HIF-1α polyclonal antibody (1:100; Novus Biologicals), rabbit anti–Iba1 polyclonal antibody (1:1000, Wako) and Alexa Fluor 488®–conjugated mouse anti–glial fibrillary acidic protein (GFAP) monoclonal antibody (1:200; Cell Signaling Technology). The following secondary antibodies were used: AlexaFlour488-conjugated goat anti–rabbit IgG antibody (1:500, Invitrogen) and goat anti–rabbit immunoglobulins conjugated to peroxidase-labeled dextran polymer (Dako Envision+, Dako). For HIF-1α staining, reaction products were visualized with 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories). The relative optical densities of HIF-1α immunoreactivity were quantified using ImageJ. The fluorescently labeled sections were examined using an LSM 510 confocal microscope (Zeiss).

Western blot analysis was performed as previously described⁴² . Samples were lysed with NP-40 buffer [PBS, 01% TritonX, 05% sodium deoxycholate, 01% sodium dodecyl sulfate (SDS), 50 mM Trix-HCl and 150 mM NaCl, pH 80] containing protease inhibitors (20 mg/ml aprotinin and 1 mM phenylmethylsulfonyl fluoride), 2-mercaptoethanol and 1 mM sodium orthovanadate. Equal concentrations of protein were resolved on 10% SDS-polyacrylamide gels, and then transferred onto Poly-vinylidene Difluoride (PVDF) membranes (ATTO, Tokyo, Japan). The blots were incubated at 4 °C overnight with one of the following primary antibodies: rabbit anti-HIF-1α polyclonal antibody (1:500; Novus Biologicals), rabbit anti–HPRT monoclonal antibody (1:2000; abcam), rabbit anti-BDNF monoclonal antibody (1:500; abcam), mouse anti-β-actin monoclonal antibody (1:10000; Sigma-Aldrich). The blots were subsequently incubated with the appropriate horseradish peroxidase–conjugated secondary antibodies for 90 min and visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA, USA). The image of each band was captured and analyzed using Image Gauge (Fuji Film, Japan) and ImageJ.