Tecnai 10 electron microscope

Treated cells were washed and fixed for 30 min in 2.5% glutaraldehyde. The samples were then treated with 1.5% osmium tetroxide, dehydrated with acetone and embedded in Durcupan resin. Thin sections were poststained with lead citrate and examined in the TECNAI 10 electron microscope (Philips, Eindhoven, Netherlands) at 60 kV.

Retinal organoids (300 days of age) were washed in 1× PBS three times at 10 min intervals and fixed with Karnovsky’s fixative (4% PFA/2% glutaraldehyde in 0.1 M PBS, pH 7.4) overnight at 4°C. For TEM, organoids were washed in 1× PBS and stained in 1% osmium tetroxide (19180, Electron Microscopy Sciences, USA; made in distilled water) for 1 hr at room temperature followed by staining with 2% uranyl acetate (22400, Electron Microscopy Sciences, USA; made in distilled water). After three washes in distilled water, organoids were then dehydrated in a graded series of ice-cold ethanol (50%, 70%, 95%, and 100%) for 20 min each. Organoids were incubated in propylene oxide (00235-1, Polysciences Inc, USA) twice for 5 min, followed by a 5 min incubation in 1:1 mix of propylene oxide: Epon 812 (13940, Electron Microscopy Sciences, USA). Organoids were allowed to infiltrate in Epon 812 overnight at room temperature. Next day, the organoids were embedded in PELCO silicone rubber molds (105, Ted Pella Inc, USA) using Epon 812, and polymerized for 48 hr at 60°C. Ultrathin (70 nm) sections were collected and imaged using a Philips Tecnai 10 electron microscope at the VA Medical Center, San Francisco, USA.

D180 retinal organoids (hiPSC CRX^WT control, CRX^T155ins4/+, and CRX^K88Q/+) were fixed overnight in 4% PFA (Fisher Acros, 41678-500) + 2.5% glutaraldehyde (Polysciences, 00376) in 0.1 M PO₄ buffer solution and rinsed four or five times with 0.1 M PO₄ buffer. Fixed organoids were then stained in 1% osmium tetroxide (OsO₄; Electron Microscopy Sciences, 19180) in distilled H₂O (dH₂O) for 1 h at room temperature. Organoids were washed in five changes of cold dH₂O, followed by staining in 2% uranyl acetate (Electron Microscopy Sciences, 22400) in dH₂O for 1 h at 37°C. After washing in dH₂O, the organoids were dehydrated in ethanol (50%, 70%, 95%, 2 × 100%), for 20 min each. The dehydration process was continued with five 20-min incubations in absolute ethanol. The organoids were exposed to propylene oxide (PO; Polysciences Inc., 00235-1) for two 5-min rinses, and then infiltrated with a 1:1 ratio of Epon resin (Epon-812; Electron Microscopy Sciences, 13940) and PO overnight at room temperature. The next day, the mixture was replaced with degassed 100% Epon resin for 1–2 h. The organoids were then embedded in 100% Epon resin in PELCO Silicone Rubber Molds (Ted Pella Inc., PELCO 105) via polymerization for 48 h at 60°C. Sections 70 nm thick were collected on copper mesh grids and imaged using a Philips Tecnai 10 electron microscope.