Anti sox9 sry box transcription factor 9

Eyes were enucleated and fixed in 4% paraformaldehyde overnight at 4°C. After a single PBS wash, eyes were cryoprotected in 30% sucrose overnight at 4°C and embedded in optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA) and cryosectioned into 15-μm thin sections. Sections were fixed in dry ice-cold acetone for 15 min and washed with PBS three times. Antigen retrieval was then performed by incubating the sections using the buffer containing 0.1mtris.HCL ph 8, 50 mM EDTA ph 8.0 and 20 μg/ml proteinase K for 10 min at room temperature. After washing two times in PBS, retinal sections were blocked with animal-free blocker (Vector Laboratories, Burlingame, CA, USA) containing 0.5% Triton X-100 for 1 h at room temperature, then primary antibodies anti-GFAP (Glial fibrillary acidic protein) (NeuroMab, Davis, CA, USA), anti-Sox9 (SRY-Box Transcription Factor 9) (EMD Millipore, Billerica, MA, USA) were applied and incubated at 4°C overnight in a moist chamber. Sections were washed three times with PBS with 0.1% triton X 100 and incubated with secondary antibodies (Invitrogen; Jackson Immuno Research, West Grove, PA, USA) for 1 h at room temperature. Sections were mounted with hard-set mounting media containing DAPI (Vector labs) and fluorescent microscopy was performed. One eye from each mouse was included in this analysis.