Immunostaining and Microscopy
Zebrafish and rat samples for immunohistochemistry (unfixed and fixed in 4% paraformaldehyde) were rinsed in 30% sucrose in PBS and directly frozen in Tissue Tek in melting isopentane. Cryosections of unfixed adult zebrafish and rat eyes (7 μm) were stained for Poc1b with anti-hsPOC1B (1:50; generously provided by Chad Pearson and Mary Pinter, University of Colorado) as described for cultured cells by Pearson et al.15 Sections were counterstained with GT335 (1:100; mouse monoclonal antibody against polyglutamylated tubulin, kindly provided by Carsten Janke, Centre National de la Recherche Scientifique Centre de Recherches en Biochimie Macromoleculaire). Sections of fixed zebrafish morphants were stained for green and red double cones with a mouse monoclonal antibody (zpr-1, raised against Arr3a, 1:500; ZIRC) and for rods with anti-rhodopsin (1:500; Novus Biologicals). Sections were washed with PBS, permeabilized for 20 min in 0.01% (v/v) Tween-20 in PBS, and washed again. Next, sections were blocked with 10% normal goat serum and 2% BSA in PBS, and primary antibodies were incubated overnight at 4°C in blocking buffer. After washing with PBS, secondary antibodies were incubated in blocking buffer for 45 min at room temperature. Samples were counterstained with DAPI and mounted with Prolong Gold (Life Technologies). For all sections, goat anti-mouse or goat anti-rabbit (Alexa 488 or 568, respectively, 1:500; Life Technologies) secondary antibody was used.