Cryopreservation and Immunostaining of Retinal Organoids

The AON-treated ROs were fixed in 2% paraformaldehyde (Thermo Fisher Scientific) and 5% sucrose (Thermo Fisher Scientific) for 15 min at 4°C, followed by a 30-min incubation in 7.5% sucrose, 30 min in 15% sucrose, and 2 h incubation in 30% sucrose. The organoids were transferred to a cryomold and embedded in 7.5% gelatin (porcine skin; Merck KGaA) and 10% sucrose. The sample blocks were then frozen at −80°C. Sections of 10 μm thickness were sliced on a Cryotome FSE (Thermo Fisher Scientific), rehydrated in PBS, and stained following the protocol described by Cowan et al.⁴⁶ (link) ABCA4 was detected using the anti-ABCA4 3F4 clone (1:100; Abcam, Cambridge, UK), rhodopsin was stained using the anti-rhodopsin 4D2 clone (1:300, Invitrogen), mitochondria were detected with an anti-MTCO2 antibody (1:150; Abcam), and nuclei were stained with Hoechst 33,342 (1:1000; Thermo Fisher Scientific). Images were collected on an LSM 800 confocal microscope (Carl Zeiss, Oberkochen, Germany) using a 60× objective and analyzed with ZEN Blue edition (Carl Zeiss) using the maximum intensity projection.