Mouse antibody to tuj1

For immunostaining, human NSCs or hiPSCs were seeded in 24‐well culture dishes with a 1 × 1 cm diameter glass coverslip in each well. After infection or treatment, cells were fixed with 4% paraformaldehyde in PBS at 4°C for 10 minutes and permeabilized with 0.3% Triton X‐100 in PBS for 15 minutes before being treated with 5% bovine serum albumin (BSA) for 1 hour at RT. Cells were incubated overnight at 4°C with various combinations of the following primary antibodies: rabbit antibody to Ki67 (Abcam, 1:500), rabbit antibody to Nestin (Millipore, 1:500), mouse antibody to Tuj1 (Covance, 1:500), goat antibody to Sox2 (R&D, 1:300), rabbit antibody to Oct4(Abcam, 1:500), or goat antibody to Sox1(R&D, 1:300). After washing with PBS/1% BSA three times, cells were incubated with Alexa Fluor 594‐labelled donkey anti‐goat IgG, Alexa Fluor 647‐labelled donkey anti‐rabbit IgG or Alexa Fluor 488‐labelled donkey anti‐mouse secondary antibodies in the dark for 1 hour. Followed by washing with PBS/1% BSA, cells were stained with DAPI (Sigma) and mounted on slides. Images were acquired under microscope (LAS SP8; Leica, FV10‐ASW 4.2; Olympus, Zeiss Z1 and Zeiss Z.1 Lightsheet microscope; Zeiss). A Canny Edge Detection plugin of ImageJ was used to measure fold density in images of Hoechst‐stained organoids by stereoscopic microscope.