Rabbit anti vimentin antiserum

After 6 days in culture, the cells were fixed in cold methanol and washed with PBS (phosphate buffered saline, pH 7.0). After blocking non-specific antigens with blocking buffer (3% BSA and 0.1% Triton X-100 in PBS), the cells were incubated with the following primary antibodies at a dilution of 1:2,000: a mouse anti-βIII-tubulin antibody (Promega Madison, WI, USA) as a specific RGC marker and a rabbit anti-Vimentin antiserum (Abcam, Cambridge, England) as a specific marker of Müller cells. After washing the cells, antibody binding was detected with an anti-mouse Alexa Fluor 488 and an anti-rabbit Alexa Fluor 555 (Life Technologies, Carlsbad, AC, USA) secondary antibodies, diluted 1:1,000. In addition, the cell nuclei were labeled with DAPI (Life Technologies, Carlsbad, AC, USA) at a dilution of 1:10,000.

After the sections were scanned they were washed with methanol at room temperature (RT) for 5 min to remove the DAN and they were then fixed with 4% PFA (paraformaldehyde) at RT for 2 min. The sections were immunostained as described previously^{38 (link)} and after washing twice in PBS-Triton X-100 for 10 min, they were incubated overnight with the primary antibodies (diluted 1:2000): a mouse anti-βIII-tubulin antibody (Promega Madison, WI, USA) as a specific RGC marker; and a rabbit anti-Vimentin antiserum (Abcam, Cambridge, England) as a specific marker of Müller glia. After washing twice in PBS, antibody binding was detected for 1 h with an Alexa Fluor 555 conjugated goat anti-mouse antibody (Invitrogen, Eugene, Oregon, USA) and an Alexa Fluor 488 conjugated goat anti-rabbit antibody (Invitrogen, Eugene, Oregon, USA), both diluted 1:1000 in PBS-BSA (1%). Finally, the sections were washed twice with PBS for 10 min and mounted with a coverslip in PBS-Glycerol (1:1).