Mice were euthanized with intraperitoneal injection of Nembutal, and eye cups were fixed in 4% paraformaldehyde. Tissue was cryoprotected in sucrose, frozen, and sectioned at 20μm in a cryostat. Slides were incubated successively with blocking solution, primary antibodies (12h-16h at 4′C) and AlexaFluor-confugated secondary antibodies (Invitrogen; 3h at room temperature). Primary antibodies were: anti-GFP (Aves and Chemicon); anti-Calbindin (Swant); anti-choline acetyltransferase (Chemicon); anti-protein kinase Cα (AbCam); anti-neurokinin receptor 3 (Calbiochem); anti-synaptotagminII (Zebrafish International Resource Center); anti-Disabled-1 (gift from T.Curran); anti-Gγ13 (Santa Cruz); anti-Bassoon (Stressgen); anti-synaptophysin (Zymed); anti-Chx10 (Exalpha Biologicals); anti-Sox9 (Chemicon); anti-glutamine synthetase (BD Biosciences); anti-cleaved Caspase-3 (Cell Signaling Technology); anti-Brn-3a (Chemicon); anti-VGlut3 (Chemicon); anti-syntaxin (Sigma); anti-Thy1.2 (BD Pharmingen); anti-GlyT1 (Santa Cruz); and anti-tyrosine hydroxylase (Chemicon). Peanut agglutinin was from Invitrogen. Nuclei were labeled with DAPI, Po-pro1, or NeuroTrace Nissl 435/455 (Invitrogen).
For measurements of retinal layer thickness and cell number, areas were chosen at equivalent retinal eccentricities from the optic nerve head or ora serrata. Layer thickness was measured on single optical sections, adjacent to the optic nerve head. Two to four areas were measured from each retina and two sets of perpendicular measurements were made per area. Both Chx10-Cre; Pcdh-γfcon3/+ and Pcdh-γfcon3/+ littermates were used as controls for Chx10-Cre;Pcdh-γfcon3/fcon3 mutants, and similarly for Pcdh-γfdel. Immunolabeled cells were quantified from 0.13 mm2 (calbindin, ChAT, Brn3a, and Paxα-GFP), 0.05 mm2 (Chx10), 0.02 mm2 (Sox9) or 1280 μm2 (photoreceptors) optical sections. Apoptotic cells were counted on sections spanning the optic nerve head to the ora serrata. Cells were classified as apoptotic if cleaved caspase-3 immunoreactivity partially or completely surrounded a nucleus. Means were compared using ANOVA, Student's t test on condition of equivalent variances determined by F-test, or with Mann-Whitney non-parametric test.
In situ hybridization of retinal sections was performed as described previously (Wang et al., 2002 (link)).
Retinas were dissociated with papain by a modification of the protocol described by Meyer-Franke et al (Meyer-Franke et al., 1995 (link)). Dissociated cells were plated onto poly-D-lysine coated 8-well Permanox chamber slides (Nunc), then fixed with 4% paraformaldehyde/4% sucrose for 15 minutes, and immunostained. RGCs were enriched with CD90 magnetic Microbeads (Miltenyi-Biotec).
For measurements of retinal layer thickness and cell number, areas were chosen at equivalent retinal eccentricities from the optic nerve head or ora serrata. Layer thickness was measured on single optical sections, adjacent to the optic nerve head. Two to four areas were measured from each retina and two sets of perpendicular measurements were made per area. Both Chx10-Cre; Pcdh-γfcon3/+ and Pcdh-γfcon3/+ littermates were used as controls for Chx10-Cre;Pcdh-γfcon3/fcon3 mutants, and similarly for Pcdh-γfdel. Immunolabeled cells were quantified from 0.13 mm2 (calbindin, ChAT, Brn3a, and Paxα-GFP), 0.05 mm2 (Chx10), 0.02 mm2 (Sox9) or 1280 μm2 (photoreceptors) optical sections. Apoptotic cells were counted on sections spanning the optic nerve head to the ora serrata. Cells were classified as apoptotic if cleaved caspase-3 immunoreactivity partially or completely surrounded a nucleus. Means were compared using ANOVA, Student's t test on condition of equivalent variances determined by F-test, or with Mann-Whitney non-parametric test.
In situ hybridization of retinal sections was performed as described previously (Wang et al., 2002 (link)).
Retinas were dissociated with papain by a modification of the protocol described by Meyer-Franke et al (Meyer-Franke et al., 1995 (link)). Dissociated cells were plated onto poly-D-lysine coated 8-well Permanox chamber slides (Nunc), then fixed with 4% paraformaldehyde/4% sucrose for 15 minutes, and immunostained. RGCs were enriched with CD90 magnetic Microbeads (Miltenyi-Biotec).
