Axio image m1

Immunostaining of frozen sections was done as described previously^{18 (link),17 (link)}. Briefly, retinal explants were fixed with 4% paraformaldehyde and treated with 15% and 30% sucrose in this order, and embedded in the optimal cutting temperature compound (Sakura Fineteck). Primarily antibodies used were monoclonal antibodies against active Caspase 3 (Promega), Ki67 (BD Bioscience), HuC/D (Molecular Probes), Chx10 (Exalha Biologicals), NR2E3 (photoreceptor-specific nuclear receptor, PNR) (ppmx), glutamine synthetase (GS) (Millipore), PKC (Merck Millipore), Rhodopsin (Rho4D2, kindly donated by Dr R. S. Molday, The University of British Columbia), and a polyclonal antibody against GFP (Clontech). Nuclei were counterstained with 4′,6-diamidino-2-phnylindole, dihydrochloride (DAPI). Sections were then treated with Alexa-488-, Alexa-594-, or Alexa-680-conjugated appropriate secondary antibodies. Photos were taken under observation using Zeiss Axio Image M1 and Axio Image M2.

For a histological visualization, part of each freshly dissected tissue sample was fixed for 3 days at 4°C in a 4% paraformaldehyde solution in 0.01 M phosphate buffer (pH 7.4), routinely embedded in paraffin, and sectioned in 4–5-µm thick slices employing a rotatory microtome Microm HM350S (Germany). Tissue sections were stuck on microscope slides by electrostatic attraction (SuperFrost Ultra Plius, Thermo Scientific, Braunschweig, Germany) and dried up to 12 h at 50°C. Staining with Masson’s trichrome (Sigma-Aldrich, Germany) and hematoxylin-eosin (Sigma-Aldrich, Germany) was used to characterize LSCC. The preparations were examined with the fluorescent microscope AxioImage M1 (Zeiss, Gottingen, Germany). Bright field images were taken using EC Plan-Neofluar 10x/0.3, Plan-Apochromat 20x/0.8, EC Plan-Neofluar 40x/0.75, or Plan-Apochromat 63x/1.40 OI lens and the high-resolution color camera AxioCam HRc (Zeiss, Gottingen, Germany).