Cm3050 s cryostat

Pups were collected at birth [postnatal day (P) 0], and 6 days after birth (P6). They were transcardially perfused at a speed of 1 mL/min (P0) and 2 mL/min (P6) with phosphate-buffered saline (PBS) for 5 min, followed by 4% paraformaldehyde (PFA) in PBS for 10 min. Pups were decapitated and heads further fixed in 4% PFA/PBS overnight. The following day, heads were washed with PBS for 1 h, and then brains removed from skulls. Brains were then transferred to 15% sucrose for 12 h at 4°C, then to 30% sucrose for another 12 h at 4°C, and finally to OCT compound (Fisher Healthcare) for 45 min at room temperature (RT) to be cryoprotectively frozen. Freezing took place submerged in OCT-filled polyethylene molds set in dry ice-chilled methanol. Embedded brains were stored at −80°C until use and sectioned on a Leica CM3050S cryostat, collecting 10 μm sections at every 100 μm on Superfrost Plus glass slides (Thermo Fisher Scientific). Sections were left to dry on a Premiere Slide Warmer XH-2004 overnight and, the following day, kept at −80°C until ready to be further processed. Embryo collection took place at embryonic day (E) 14.5. After transcardial perfusion, embryonic brains were collected and fixed in 4% PFA/PBS for 3 h. Subsequently, brains were cryoprotected and then frozen following the previously described steps.

Eyes from euthanized C57BL/6J mice were enucleated, washed in PBS, and either fixed in 4% paraformaldehyde (PFA) for immunofluorescence (IF) or dissected to isolate the retina for preparation of RNA and/or protein. For IF analysis, enucleated eyes were washed and fixed in 4% PFA, incubated in ascending series of sucrose, and frozen in a sucrose OCT solution as previously described [11 (link)]. Thick sections (12 μm) were cut using Leica CM3050S cryostat onto Superfrost Plus slides (ThermoScientific) and stored at −80 °C until use. Retinas from enucleated eyes were isolated as previously described [11 (link)]. Isolated retinas were immediately processed for RNA isolation using RNeasy kit (Qiagen).

Quantification of rod photoreceptor degeneration was analysed as previously described^{41 (link)}. EGFP-positive offspring from rho:EGFP crosses to wild-type fish were culled at 10 d.p.f. by the addition of an excess of MS222 and fixed overnight in 4% paraformaldehyde in PBS at 4 °C. After washes in PBS, the zebrafish were equilibrated overnight in 30% sucrose in PBS at 4 °C, embedded in Scigen Tissue Plus optimal cutting temperature compound and frozen on dry ice before being stored at −20 °C. Transverse cryosections (10 μm) were cut through the central retina using a LEICA CM3050 S cryostat and collected on Thermo Scientific Menzel Gläser, SuperFrost Plus slides. Imaging and analysis of images were performed as described^{41 (link)}.

Reconstructed human tissues were fixed in 4% paraformaldehyde overnight at 4°C. The tissue was washed in PBS three times for 15 min each. The tissue was then taken through a gradient of 15% and 30% weight/volume sucrose in PBS. Whole tissues were removed from sucrose, blotted dry, and embedded in Tissue-Tek CRYO-OCT Compound (Andwin Scientific, Tryon, NC, United States). Blocks were stored at −80°C, sectioned on a Leica CM3050 S cryostat into 12 μm thick sections, and placed on SuperFrost^TM Plus slides (Thermo Fisher Scientific).