Lsm 710 confocal laser point scanning microscope

Cells were fixed with 4% PFA and stained according to a previously described protocol (Miranda et al., 2018 (link)). Primary and secondary antibodies are listed on Supplementary Table S1. Fluorescence images were acquired either with the fluorescence optical microscope (Leica DMI 3000B) and a digital camera (Nikon DXM 1200), or with Zeiss LSM 710 Confocal Laser Point-Scanning Microscope using 20x objective with 80% digital zoom, and integrated density was calculated for each channel using ImageJ software.

Cells were fixed with 4% (v/v) paraformaldehyde (PFA; Sigma) and stained according to a previously described protocol (Miranda et al., 2015 (link)). MAP2 (Sigma, 1:500), glial fibrillary acidic protein (GFAP, Abcam, 1:200), Synaptophysin (SYN; Abcam, 1:200), ZO-1 (Novex, 1:100), SOX2 (R&D, 1:200), PAX6 (Covance, 1:400), NESTIN (R&D, 1:400), Ki-67 (Abcam, 1:100), HuC/D (Thermo Fischer Scientific, 1:100), activated CASPASE3 (pCASP3, Cell Signaling, 1:400), were used as primary antibodies whereas goat anti-mouse IgG Alexa Fluor–488 or 546 (1:500, Invitrogen), goat anti-rabbit IgG Alexa Fluor–488 or 546 (1:500, Invitrogen) were used as secondary antibodies. Fluorescence images were acquired with Zeiss LSM 710 Confocal Laser Point-Scanning Microscope using 20× and 63× objectives and integrated density were calculated for each channel using ImageJ software. The ratio between integrated density for the marker of interest and nuclear counterstaining with DAPI was calculated for each image. For each staining, the same acquisition settings were applied for all images.