Additional NotesImmunohistochemical analysis of NeuN-positive neurons.
hPSC derived retinal organoids were washed once in PBS and then fixed in 4% paraformaldehyde (PFA) for 30 min, then immersed in 20% sucrose/PBS at 4C overnight. 20% sucrose/PBS was removed completely and the samples embedded in OCT matrix and frozen in liquid nitrogen. For immunohistochemistry, sections were blocked and permeabilised with PBS containing 0.3% triton X-100, 10% serum and 1% bovine serum albumin (block/permeabilization solution) for 1 hour. Abcam's NeuN primary antibody (white) was incubated overnight at 4C. Following primary antibody incubation, sections were incubated with Alexa-Fluor secondary antibody in 1:500 dilutions at room temperature for 2 hours, washed in PBS and counterstained with DAPI (Blue). Images were captured with a confocal microscope.