Eyes were processed for immunohistochemistry as well as for hematoxylin and eosin staining. Immunohistochemistry was performed as described earlier by Mataruga et al.
35 In brief, eyes were enucleated and opened by an encircling cut at the limbus. The retina in the eye cup was immersion fixed for 30 minutes in 4% paraformaldehyde (PA) in 0.1 M phosphate buffer (PB) at room temperature and washed in PB several times. Tissue was incubated in 10% sucrose/PB for 1 hour, followed by 30% sucrose/PB overnight. The retina was flat embedded and frozen in optimal cutting temperature compound (NEG-50, Richard-Allan Scientific; Thermo Fisher Scientific, Germany). Vertical sections (20-μm thickness) were cut on a cryostat (HM 560 CryoStar; Microm, Walldorf, Germany) and collected on Superfrost Plus slides (Menzel, Braunschweig, Germany). Sections were pretreated with blocking solution (5% Chemiblocker [Chemicon, Hofheim, Germany], 0.5% Triton X-100 in PB, and 0.05% NaN
3) for 1 hour, followed by incubation with primary antibodies overnight, and diluted in the same solution. Primary antibodies directed against the following antigens were used: antiglial fibrillary acidic protein (GFAP, raised in chicken, 1:2000; Novus Biologicals, Cambridge, UK), glutamine synthetase (raised in mouse, 1:4000; BD Biosciences), anti-calbindin 28K (CabP; raised in mouse, 1:1000; Sigma-Aldrich), anti-protein kinase C α (PKCa; raised in rabbit, 1:4000; Santa Cruz, Heidelberg, Germany), calretinin (AB1550, raised in goat, 1:3000; Millipore, Schwalbach, Germany), HCN1 (RTQ-7C3, raised in rat, 1:10; F. Müller, Forschungszentrum Jülich GmbH, Jülich, Germany), recoverin (AB5585, raised in rabbit, 1:2000; Millipore), and rhodopsin (1D4, 1:500; R.S. Molday, British Columbia, Canada). Sections were washed in PB and incubated in secondary antibodies diluted in 5% Chemiblocker (Chemicon), 0.5% Triton X-100 in PB for 1 hour, washed in PB, and coverslipped with Aqua Polymount (Polysciences, Eppelheim, Germany). Secondary antibodies included donkey anti-chicken Cy2 (1:400; Dianova, Hamburg, Germany), donkey anti-mouse Cy3 (1:100; Dianova), donkey anti-rabbit Cy2 (1:400; Dianova), donkey anti-goat Alexa 647 (1:200; Invitrogen, Germany), donkey anti-rat Cy3 (1:500; Dianova), and donkey anti-mouse Dy649 (1:500; Dianova). The lectin peanut agglutinin (PNA, biotinylated, 1:1600; Sigma-Aldrich) was visualized using streptavidin Alexa 647 (1:100; Invitrogen, Darmstadt, Germany). Sections were examined with a Leica TCS confocal laser scanning microscope (Leica Microsystems, Heidelberg, Germany) with ×63/1.4 oil immersion lenses. Images were processed and printed with Adobe Photoshop (Adobe Systems, München, Germany). For triple labeling, primary antibodies were mixed and applied simultaneously. All secondary antibodies were highly cross-absorbed and were carefully tested to exclude reactions with the wrong primary antibody. Concentration of the antibodies, laser intensity, and filter settings were carefully controlled; and the sequential scanning mode was employed to completely rule out cross-talk between the fluorescence detection channels. Band pass filters of 500 to 530 nm for green fluorescence (Cy2), 580 to 650 nm for red fluorescence (Cy3), and 680 to 750 nm for infrared fluorescence (Alexa 647, Dy649) were used.