Ab73593

For immunofluorescence labeling, liver sections were (co-)stained with antibodies directed against GS (1:1000; ab73593, Abcam), SOX9 (1/100; AB5535, Millipore), F4/80 (1:100; MCA497RT, BioRad), HNF4ɑ (1/100; sc6556, Santa Cruz) or CD31 (1:200; MEC13.3, BioLegend). Secondary antibodies were either anti-rabbit AlexaFluor 488; or anti-rabbit, anti-goat or anti-rat AlexaFluor 647 (1:1000, Invitrogen). Hoechst (1:10 000, 62249, Thermo Scientific) was used to counterstain the nuclei. Liver sections were imaged using the Zeiss AxioImager.Z2 microscope.

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.

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).

The eyes were enucleated, fixed with 4% PFA for 30 min at room temperature, and immersed in OCT compound. Then, they were frozen and cut into 8-μm sections using a cryostat (CM1800, Leica Microsystems, Wetzlar, Germany) and mounted onto MAS-coated glass slides (MAS-01, Matsunami, Osaka, Japan). After removing the OCT compound in PBS containing 0.3% Triton X-100 for 15 min, the sections were blocked with 10% NGS in PBS for 1 h. Then, they were incubated with primary antibodies (chicken anti-vimentin [ab24525, Abcam], mouse anti-Rho [1D4, Abcam], FITC-conjugated peanut agglutinin (PNA) [L7381, Sigma-Aldrich], rabbit anti-PSD95 [monoclonal, 1/100, D27E11, Cell Signaling Technology, Danvers, MA, USA], rabbit anti-GS [ab73593, Abcam]) diluted in PBS at 4°C overnight. After washing with PBS, the sections were incubated with Alexa-conjugated secondary antibody (Goat anti-Rabbit IgG [H+L], Alexa Fluor™ 488, A-11008, Goat anti-Rabbit IgG [H+L], Alexa Fluor™ 546, A-11035, Goat anti-Mouse IgG [H+L], Alexa Fluor™ 647, A-21235) diluted in PBS for 1 h at room temperature, and the nuclei were counterstained with DAPI. Images were acquired using a BZ-X710 confocal microscope.

After fixation with 4% PFA for 30 min at room temperature, the eyes of mice were embedded in paraffin and sliced into 3-μm sections. After removing the paraffin, the sections were activated, blocked, and incubated with primary antibodies (chicken anti-vimentin [ab24525, Abcam], mouse anti-Rho [1D4, Abcam], FITC-conjugated PNA [L7381, Sigma-Aldrich], rabbit anti-PSD95 [D27E11, Cell Signaling Technology, rabbit anti-GS [ab73593, Abcam]) diluted in PBS at 4°C overnight. After washing with PBS, the sections were incubated with Alexa-conjugated secondary antibody (Goat anti-Chicken IgY, [H+L], Alexa Fluor™ 488, A-11039, Goat anti-Rabbit IgG [H+L], Alexa Fluor™ 488, A-11008, Goat anti-Rabbit IgG [H+L], Alexa Fluor™ 546, A-11035, Goat anti-Mouse IgG [H+L], Alexa Fluor™ 647, A-21235) diluted in PBS for 1 h at room temperature, and the nuclei were counterstained with DAPI. Images were acquired using a BZ-X710 confocal microscope. The Rho-positive length in the outer retinal layers was analyzed using ImageJ software. At least three sections, including the optic nerve, were measured per eye, and the average was calculated.