Ab5417

Sections of samples embedded in paraffin were deparaffinized in a xylene ethanol series, placed in Tris-EDTA buffer for antigen retrieval (10 mM Tris, 1 mM EDTA, 0.05% Tween, pH = 9.0), and then blocked in 5% bovine serum albumin. Sections were immunostained for rhodopsin (rod photoreceptors) (Ab 5417; Abcam, United Kingdom; diluted 1:100). Detection of the primary antibodies was performed using fluorescein isothiocyanate (FITC)–conjugated goat anti-mouse IgG secondary antibody (F-2761; Thermo Fisher Scientific, United States; diluted 1:400). Nuclei were detected using 4′,6′-diamino-2-phenyl inodole (DAPI), which was included in the mounting solution (Solarbio; Beijing, China).

Cultured cells were fixed using 4% paraformaldehyde (PFA) at room temperature for 20 min. After washing with PBS, cells were incubated with 0.3% Triton X-100 (FUJIFILM Wako) in PBS for 15 min and blocked with PBS containing 10% normal goat serum (NGS) (Thermo Fisher Scientific) for 1 h at room temperature. Cells were then incubated with primary antibodies (chicken anti-vimentin [polyclonal, 1/1000, ab24525, Abcam, Cambridge, UK], rabbit anti-glutamine synthetase [GS] [polyclonal, 1/100, ab73593, Abcam], mouse anti-rhodopsin [Rho] [monoclonal, 1/100, ab5417, Abcam], and rat anti-mouse CD44-PE [monoclonal, 1/100, 1M7, eBioscience, San Diego, CA, USA]) diluted in PBS at 4°C overnight. Next, the cells were incubated with Alexa-conjugated secondary antibodies (Goat anti-Chicken IgY, [H+L], Alexa Fluor™ 488, A-11039, Goat anti-Rabbit IgG [H+L], Alexa Fluor™ 546, A-11035, Goat anti-Mouse IgG [H+L], Alexa Fluor™ 647, A-21235, Thermo Fisher Scientific) diluted in PBS for 1 h at room temperature, and the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Immunofluorescence images were acquired using a BZ-X710 confocal microscope (Keyence, Osaka, Japan). For cell counting, at least three fields were selected per dish.

In situ hybridization, plastic sectioning, and immunolabeling of cryosections were performed as described previously^{35 (link)}. Immunolabeling of whole retinas was conducted, using dissected optic cups of para-formaldehyde (PFA)-fixed embryos. zpr1 antibody (ZIRC, Eugene, Oregon; 1:100), anti-zebrafish green opsin (1:500)^{20 (link)}, anti-red opsin (1D4, abcam ab5417, 1:200–500)^{17 (link)}, anti-PCNA antibody (clone PC10, Sigma P8825; 1:200) were used. TUNEL was performed using an In Situ Cell Death Detection Kit (Roche). Rhodamine-conjugated phalloidin (Invitrogen, R415) at 0.66 μM was applied to visualize actin filaments on cryosections labelled with zpr1 antibody. Images were scanned using a confocal laser scanning microscope (Carl Zeiss, LSM510 and LSM710).

Immunocytochemical analysis was performed as previously described. Briefly, the cultured cells or neurospheres were fixed in 4% paraformaldehyde-PBS for 30 min, blocked in PBS containing 5% goat serum and 0.3% TritonX-100 at 37 °C for 1 h, and then incubated with one of the following primary antibodies overnight at 4 °C: rabbit anti-GS (1:100, Abcam, ab73593), mouse anti-vimentin (1:75, Abcam, ab8976), rabbit anti-Sox2 (1:100, Abcam, ab92494), mouse anti-nestin (1:100, Abcam, ab6320), mouse anti-rhodopsin (1:50, Abcam, ab5417). Following PBS wash, cells or neurospheres were incubated in fluorophore-conjugated goat anti-mouse IgG (ZSGB-BIO, China) or goat anti-rabbit IgG (Multi Sciences, China) for 2 hours in the dark and then counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma, USA) for five minutes. Fluorescent images were recorded using confocal microscopy (Leica SP8, Germany) or fluorescent microscopy (Leica DM5000B, Germany).