Eyes were fixed in 4% paraformaldehyde for 1 h before cryoprotecting in 20% sucrose and embedding in OCT (RA Lamb) and freezing in isopentane precooled in liquid nitrogen. Cryosections were cut at 18-μm thick and all sections were collected for analysis. For histology and cell counting, cryosections were air-dried for 30 min and washed in PBS containing Hoechst 33342 (10 μM) for 5 min before mounting. For immunohistochemistry, sections were blocked in 5% goat serum and 1% bovine serum albumin and triton X-100 (0.05% in PBS at RT for 1 h. Primary antibody was incubated overnight at 4 °C. After washing, sections were incubated with secondary antibody for 2 h at RT, washed and counter-stained with Hoechst 33342 (10 μM). Primary antibodies used included: LaminB antibody (mouse monoclonal; Abcam Ab8980; 1:200); rod α-transducin (rabbit polyclonal; Santa Cruz SC389; 1:1,000); CRE Recombinase (Mouse monoclonal; Millipore MAB3120; 1:200). Alexa fluor 488, 546 and 633 secondary antibodies (Invitrogen-Molecular Probes) were used at a 1:500 dilution. Negative controls omitted the primary antibody. Retinae receiving rEGFP were examined both unstained, as is typical for assessing retinae receiving retinal transplants9 (link)12 (link) and following immunostaining with a directly conjugated chicken anti-GFP antibody (Invitrogen-Molecular Probes; 1:200 for 2 h before mounting).
Free full text: Click here
