Cfi apo lwd lambda s 40 objective

Cerebral organoid slices were fixed in 4% PFA for 20 minutes. Slices were blocked and permeabilized in 5% normal donkey serum 0.25% Triton X-100 for 1 hour at room temperature. Primary and secondary antibody mix was prepared in 5% normal donkey serum 0.1% Triton X-100 and incubated overnight at 4 °C. Samples were mounted in VECTASHIELD HardSet (Vector Laboratories) and mosaics (3 × 3 tiles) were acquired with CFI Apo LWD Lambda S ×40 objective (NA 1.15; WD 0.61–0.59, Nikon). Primary antibodies used included: sheep anti-EOMES (1:200, R&D Systems AF6166), chicken anti-GFP (1:500, Abcam Ab13970), mouse anti-Sox2 (1:500, Abcam Ab79351) and rabbit anti-NeuroD2 (1:500, Abcam Ab104430).

Human fetal brain and cerebral organoid slices in culture were fixed in 4% paraformaldehyde (PFA) for 2 hours. Slices were boiled in sodium citrate buffer (10 mM, pH 6) for 20 minutes and cooled down at room temperature (antigen retrieval). Slices were then blocked in phosphate-buffered saline (PBS)-Triton 100×0.3%-donkey serum 2% at room temperature for 2 hours, incubated with primary antibody overnight at 4 °C in blocking solution, washed in PBS-Tween 0.05%, and incubated with secondary antibody overnight at 4 °C in blocking solution before final wash and mounting in Aquapolymount. Mosaics (tile scans) of fixed tissue were acquired with a CFI Apo LWD Lambda S ×40 objective (WI NA 1.15 WD 0.61–0.59, Nikon).