Ab15282

Sections were incubated overnight at 4 °C with the primary antibodies (rhodopsin: Sigma-Aldrich, MAB5316, 1:300; Iba1: Fujifilm Wako Chemicals, 019-19741, 1:200; GFAP: Merck Millipore, G3893, 1:500; and cone-arrestin: AB15282, Merck Chemicals GmbH, 1:1000) followed by incubation with secondary antibodies (Alexa Fluor™ 568 dye-conjugated goat anti-mouse IgG, ThermoFischer, Waltham, MA, USA, A 11031 or Alexa Fluor™ 488 dye-conjugated goat anti-rabbit IgG, Cell Signaling Technology, Danvers, MA, USA, A 11034, 1:500). Negative controls were carried out by omitting the primary antibody.

Eye cryosections were re-fixed with formaldehyde 3.7% (w:v) in 200 mM HEPES buffer, pH 7.0 for 15 min, followed by 3 5-min PBS washes with agitation at room temperature. Retinal sections were permeabilized with 1% (v:v) Triton X-100 (Sigma-Aldrich, T8787), in PBS and then blocked for 1 h with BGT (3 mg/mL BSA [Roche, 10735108001], 0.25% Triton X-100, 100 mM Glycine [VWR Chemicals, 0167] in PBS). Primary antibodies were incubated in BGT overnight at 4ºC. Secondary antibodies were incubated for 1 h at room-temperature in BGT and darkness. Nuclei were stained with DAPI (1 μg/mL) and cryosections were mounted with Vectashield. Antibodies used were: CALB1 (Sigma-Aldrich, C2724), PRKCA (Sigma-Aldrich, P4334), SNCG (Abnova, H00006623-M01), AIF1 (Wako, 019-19741), ARR3 (EMD Millipore, AB15282), GLUL (EMD Millipore, MAB302), SAG (Santa Cruz Biotechnology, sc-166383,), and GFAP (Dako, Z0334).

Microglial cells were detected using a rabbit monoclonal anti-Iba1 antibody (1:1000–500; ab178846: Abcam, Cambridge, UK). Astrocytes and Müller cells were detected with a goat monoclonal anti- Glial fibrillary acidic protein (GFAP) antibody (1:500; 019-19741: Abcam, Cambridge, UK). The synaptic connections were detected with a mouse monoclonal anti-Bassoon antibody (1:750; ADI-VAM-PS003; Enzo life Science, Lausen, Switzerland). The L-cones, S-cones and rods outer segments were detected using a rabbit monoclonal anti-L/M-opsin (1:1200; ab5405; Chemicon-Millipore Iberica, Madrid, Spain), a goat monoclonal anti-S-opsin (1:1000; N-20; anti-OPN1SW; Santa Cruz Biotechnology, Heidelberg, Germany) and a mouse monoclonal anti-rhodopsin (1:1200, 1D4; Sigma-Aldrich, Madrid, Spain) antibody, respectively. Cone photoreceptors were detected using a rabbit monoclonal anti-cone arrestin antibody (1:1000; AB15282, Merck, Germany). Signs of Oxidative stress were detected with a monoclonal antibody against mouse α-8-hydroxy-2′-deoxyguanosine (8-OhdG; 1:1000, sc-66036; Santa Cruz Biotechnology, Heidelberg, Germany), which is a marker that binds oxidatively damaged proteins and DNA [75 (link)]. Retinal ganglion cells were detected using a goat polyclonal anti-Brn3a (1:750; C-20; Santa Cruz Biotechnology).

661 W cells were fixed in 4% paraformaldehyde (PFA) for 15 minutes. Cells were permeabilized and blocked in PBS containing 4% BSA and 0.5% Triton X-100 for 1 hour at room temperature, incubated overnight with primary antibody at 4 °C, and then subjected to immunohistochemistry as previously described36 (link).
Primary antibodies were rabbit anti-opsin blue (1:500; chemicon, AB5407), rabbit anti-opsin red/green (1:500; chemicon, AB5405), rabbit anti-cone arrestin (1:500; Millipore, AB15282) and mouse anti-rhodopsin (1:10000; sigma, o4886).

The protocol for immunohistochemical assays was previously described [125 (link)]. Briefly, after carbon dioxide asphyxiation, eyes were enucleated and placed in cold methanol:acetic acid:PBS (3:1:4) or freshly prepared 4% paraformaldehyde (PFA) in PBS overnight at 4°C, as appropriate for the application. The eyes were embedded in paraffin and deparaffinized 6 μm sections were incubated with anti-rhodopsin (Millipore MAB5356, 1:200, or NeoMarkers MS-1233-R7, undiluted), anti-GFAP (DAKO Z0334, 1:200), anti-β-dystroglycan (Novocastra NCL-b-DG 1:100), anti-FKRP (Novus NBP1-74745, 1:200), anti-CTBP2 (BD Biosciences, 612044, 1:200), anti-collagen IV (Chemicon AB756P, 1:200), ezrin (Cell Signaling 3145, 1:200), anti-cone arrestin (Millipore AB15282, 1:200), or anti-α-laminin (Sigma L9393, 1:100). Antibody binding was revealed using species-specific Cy3- or Alexa Fluor488-conjugated anti-IgG (Jackson Immunoresearch and Life Technologies, 1:200) and visualized by fluorescence microscopy. For negative controls, the primary antibody was omitted.