Ab84870

Retinal organoids were briefly washed once in PBS and placed in a mixture of 4% PFA + 5% sucrose in PBS for 40 minutes. Organoids were then placed in 6.25% sucrose in PBS for 1 hour, followed by 12.5% sucrose in PBS for 30 minutes and in 25% sucrose in PBS for 1 hour. All incubation steps were performed at 4°C. Organoids were then embedded in OCT, slowly frozen on dry ice, and stored at −80°C until cryosectioning. Cryosections (7 μm thick) were collected and stored at −20°C for later analysis.
For immunohistochemistry, sections were first washed once in PBS and blocked with 10% donkey serum, 0.05% Triton X-100 in PBS for 30 minutes. Primary antibodies used were the following: anti-rabbit CEP290 (Abcam) ab84870, 1:100; anti-rabbit ARL13B (Proteintech) 1:1000; anti-mouse Pericentrin (Abcam) ab28144, 1:1000; anti-mouse Rhodopsin 4D2 (Millipore) MABN15 1:1000; anti-rabbit Opsin Antibody, Red/Green (Millipore) 1:500; anti-mouse CRX (Abnova) H00001406-M02 1:1000; anti-rabbit Recoverin (Millipore) AB5585 1:1000. Primary antibodies were incubated in the blocking solution diluted 50% in PBS for 1 hour. Sections were then washed in PBS and incubated with Alexa Fluor (Thermo Fisher) secondary antibodies in the diluted blocking solution for 45 minutes. Nuclei were visualized using DAPI (2 μg/mL). Samples were washed in PBS and mounted using DAKO fluorescence mounting medium (Agilent).

Punches were blocked in IF buffer (1% BSA; [Sigma-Aldrich; A9647] and 0.05% Tween-20 [Sigma-Aldrich; P9416] in 1× PBS) for 1–2 h at RT, and incubated with primary antibody diluted in IF buffer for 24 h at 4°C. Punches were washed in 1× PBS for 1–2 h and incubated with secondary antibody and DAPI (1:10,000 final dilution; Thermo Fisher Scientific; D1306) in IF buffer for 24 h at 4°C. The following primary antibodies were used for immunostaining: mouse anti-acetylated tubulin (Sigma-Aldrich; T7451) at 1:4,000, rabbit anti-β tubulin (Abcam; ab15568) at 1:250, rabbit anti-ARL13B (Abcam; ab83879) at 1:500, rabbit anti-Cep290 (Abcam; ab84870) at 1:600, and rabbit anti-Cep164 (Proteintech; 22227–1-AP) at 1:500. Secondary antibodies Alexa Fluor 488 anti-mouse (Thermo Fisher Scientific; A11029) and Alexa Fluor 555 anti-rabbit (Thermo Fisher Scientific; A21429) were used at a 1:800 dilution to label primary antibodies for 24 h. After immunostaining, the samples were expanded in deionized H₂O for 2 h at RT with deionized H₂O exchanged every 10 min and additionally overnight at 4°C. Prior to imaging, expanded punches were mounted in Rose chambers, Attofluor cell chambers, or glass-bottom Microwell dishes (MatTek; P35G-1.5-14-C).