Immunohistochemistry (neural organoids)

The organoids were fixed in 4% paraformaldehyde for 2h 0m 0s at Room temperature .

They were subsequently washed three times with KPBS and left in a 1:1 OCT:30% sucrose solution and OCT (HistoLab) mixture 2h 0m 0s.

The organoids were then transferred to a cryomold containing OCT and frozen on dry ice and stored at -80°C.

Prior to the staining protocol, the organoids were cryosectioned at a thickness of 20 μM.

For immunohistochemistry, sections were washed in PBS1X for 0h 10m 0s and then blocked and permeabilized in Blocking solution (0.3% Triton X-100 and 4% normal donkey serum in PBS1X) for at least 1h 0m 0s.

The sections were then incubated with primary antibodies in blocking solution at 4°C 1h 0m 0s.

The primary antibodies used for neural organoids characterisation were rabbit anti-PAX6 (1:300; BioLegend, Cat# 3700, RRID:AB_2242334)) and rat anti-ZO1 NB110-68140, RRID:AB_1111431(1:300; Novus)).

After incubation with the primary antibodies, the sections were incubated for 1 h with the appropriate secondary antibodies (Alexa Fluor 488, 594, 647 used at 1:400; Molecular Probes).

Finally, the sections were mounted on gelatin-coated slides and coverslipped with PVA-DABCO containing DAPI (1:1000).