Immunohistochemical analysis was performed as described previously (Hori et al., 2019; Kubo et al., 2021). In brief, the retina used for MEA recordings and isolated mice eyes was fixed with 4% PFA in PBS for 30 min at room temperature. After three-time washes, retinas were cryoprotected by 30% sucrose in PBS overnight, embedded in an OCT compound (Sakura, Japan), frozen on dry ice, and sectioned at 20 μm of thickness. Whole-mount immunostaining was performed as previously described with some modifications (Ueno et al., 2018). The retinas were gently peeled off from the sclera, fixed with 4% PFA in PBS for 30 min at room temperature, and washed three times. Retinal sections and whole retinas were soaked in blocking buffer (5% NDS, 0.1% Triton X-100, in 1x PBS) for 1–2 hr at room temperature and incubated with primary antibodies in blocking buffer 1 or 2 overnight at 4°C. The sections were washed with PBS three times and incubated with fluorescent dye-conjugated secondary antibodies and DAPI (1:1000) for more than 2 hr at room temperature or overnight at 4°C under the light-shielded condition. The specimens were observed using a laser confocal microscope (LSM900; Carl Zeiss, Germany). For BrdU staining, mice were given an intraperitoneal injection of 20 mg/kg BrdU and sacrificed 2 hr later. Retinal sections were then pretreated with 2 N HCl at 37°C for 30 min before blocking. The antibodies and dilution ratios were as follows: anti-l-afadin (ab90809, Abcam, UK, 1:100), anti-nectin1 (D146-3, MBL, Japan, 1:100), anti-nectin2 (D083-3, MBL, Japan, 1:100), anti-nectin3 (D084-3, MBL, Japan, 1:100), anti-PKCα (P5704, Sigma, USA, 1:1000, P4334, Sigma, 1:10,000), anti-SCGN (AF4878, R&D systems, USA, 1:2000), anti-Arr3 (AB15282, Millipore, USA, 1:1000), anti-Calbindin (PC253L-100, Millipore, USA, 1:200), anti-ChAT (AB144P, Millipore, USA, 1:50), anti-Bassoon (SAP7F407, Enzo, USA, 1:1000), anti-mGluR6 (current study, 1:3000), anti-PSD95 (#124 014, Synaptic Systems, Germany, 1:3000), anti-GluR5 (Grik1, gift from Steve H DeVries, 1:2000), anti-PKARIIβ (#610625, BD biosciences, USA, 1:500), anti-Calretinin (PC235L-100UCN, Millipore, USA, 1:5000), anti-EAAT5 (HPA049124, Sigma, USA, 1:100), anti-vGlut1 (AB5905, Millipore, USA, 1:6000), anti-HPC-1 (S0664, Sigma, USA, 1:10000), anti-active caspase3 (AF835, R&D Systems, USA, 1:300), anti-Chx10 (Hori et al., 2019, 1:100), anti-Otx2 (AF1979, R&D systems, USA, 1:200), anti-Glutamine synthetase (GS, MAB302, Millipore, USA, 1:500), anti-CD31 (#557355, BD Biosciences, USA, 1:100), anti-Lhx2 (sc-19344, Santa Cruz Biotechnology, USA, 1:100), anti-RBPMS (GTX118619, GeneTex, USA, 1:500), anti-Ki67 (#556003, BD Biosciences, USA, 1:100), anti-Tuj1 (#801201, BioLgend, USA, 1:500), anti-AP2α (3B5-c, DSHB, USA, 1:1000), anti-Rom1 (gift from Robert Moldey, 1:10), anti-Rhodopsin (STJ95452, ST John’s Laboratory, UK, 1:100), anti-S-opsin (sc-14363, Santa Cruz Biotechnology, USA, 1:500), anti-M-opsin (AB5405, Sigma, USA, 1:500), anti-GFP (GFP-1010, Aves labs, USA, 1:1000), anti-β-catenin (#610153, BD Biosciences, USA, 1:1000), anti-N-cadherin (#610920, BD Transduction Laboratories, USA, 1:500), anti-BrdU (#347580, BD Biosciences, USA, 1:100), and anti-phospho-histone H3S10 (#06–570, Millipore, USA, 1:2000) antibodies.
Afadin-deficient mouse retinas exhibit severe neuronal lamination defects but preserve visual functions
