Fixation, tissue processing, and immunohistochemistry were performed as previously described50 (link) using the following primary antibodies: Calbindin (Swant, CB38, rabbit, 1:3000), PAX6 (Biolegend, 901301, rabbit, 1:300), SKOR2 (Novus, NBP2–14565, rabbit, 1:100), and NEUN (Millipore, MAB377, mouse, 1:100). All sections were counterstained using Vectashield DAPI (H1000, Vector labs) which marks all nuclei.
In situ hybridization was performed using commercially available probes from Advanced Cell Diagnostics. Manufacturer recommended protocols available on the ACD webpage were used without modification. Probes used include: LMX1A (#540661), MKI67 (#591771), ATOH1 (#417861), OTX2 (#484581), and HOXB3 (custom-made). All sections were counterstained using Fast Green.
Slides processed for fluorescent IHC were imaged using Zeiss LSM Meta confocal microscope and ZEN 2009 software (Zeiss). Brightfield imaging was performed using a Nanozoomer Digital Pathology slide scanner (Hamamatsu; Bridgewater, New Jersey). Barring minor adjustments limited to contrast and brightness to the entire image, no additional image alteration was performed.
In situ hybridization was performed using commercially available probes from Advanced Cell Diagnostics. Manufacturer recommended protocols available on the ACD webpage were used without modification. Probes used include: LMX1A (#540661), MKI67 (#591771), ATOH1 (#417861), OTX2 (#484581), and HOXB3 (custom-made). All sections were counterstained using Fast Green.
Slides processed for fluorescent IHC were imaged using Zeiss LSM Meta confocal microscope and ZEN 2009 software (Zeiss). Brightfield imaging was performed using a Nanozoomer Digital Pathology slide scanner (Hamamatsu; Bridgewater, New Jersey). Barring minor adjustments limited to contrast and brightness to the entire image, no additional image alteration was performed.
