Araldite 502 embed 812

For conventional light microscopy (LM) and transmission electron microscopy (TEM), tissues were subjected to primary aldehyde fixation in 3% glutaraldehyde (in 0.1M phosphate buffer, pH 7.2; (Electron Microscopy Sciences, Hatfield, PA, USA) for 3 h at 4°C followed by postfixation in in buffered 1% osmium tetroxide. The samples were then dehydrated through a graded series of ethanol, soaked in propylene oxide and embedded in Epon‐Araldite (Araldite 502/Embed 812, Electron Microscopy Sciences).
Blocks were cut using a Leica UltraCut UCT (Leica Microsystems, Wetzlar, Germany). Semithin sections (1–2 μm) for LM were stained with toluidine blue and were examined with a LM Leitz Dialux 20 EB (Leica Microsystems). Ultrathin sections (800 Å) were stained with uranyl acetate replacement, and lead citrate (Electron Microscopy Sciences) and observed under a Zeiss EM 10 electron microscope (Zeiss, Oberkochen, Germany).

Eyes from pigmented sh1 mice (+/− or −/−) were enucleated 3 months following the AAV injection and cauterized on the temporal side of the cornea. Fixation was performed using 2% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA) and 2% paraformaldehyde (Electron Microscopy Sciences) in 0.1 M PBS overnight, rinsed in 0.1 M PBS, and dissected under a light microscope. The temporal portions of the eyecups were embedded in Araldite 502/Embed 812 (Electron Microscopy Sciences). Semi-thin (0.5 μm) sections were transversally cut on a Leica Ultramicrotome RM2235 (Leica Microsystems, Wetzlar, Germany), mounted on slides, and stained with 1% toluidine blue (Carlo Erba, Milan, Italy) with 2% borace staining (Thermo Fisher Scientific) in deionized water. Melanosomes were counted by a masked operator in a montage of the entire retinal section obtained through acquisition of overlapping fields using a Zeiss Apotome (Carl Zeiss, Oberkochen, Germany) with 100× magnification; the entire retinal section was then reconstituted on Photoshop software (Adobe, Mountain View, CA). Melanosome counts and RPE measurements were performed using ImageJ software. Melanosome number was normalized over the length of the RPE divided by 100 μm.