Alexa fluor 488 conjugated mouse anti glial fibrillary acidic protein monoclonal 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).

Spinal cord sections of mSOD1^G93A mice were prepared as previously described [1 (link)]. The following antibodies were used: rabbit anti-JAK2 (phospho Y1007 + Y1008) monoclonal antibody (1:200; Abcam, Cambridge, UK), rabbit anti-iNOS polyclonal antibody (1:50; BD Biosciences, Franklin Lakes, NJ, USA) and Alexa Fluor 488®-conjugated mouse anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (1:200; Cell Signaling Technology, Beverly, MA, USA). The following secondary antibodies were applied: Cy5-conjugated F(ab’) 2 fragment donkey anti-rabbit IgG (1:500; Jackson ImmunoResearch Laboratories, West Grove, PA, USA).