EdU incorporation and quantitation

Pregnant females were pulsed with EdU via IP injection when embryos were age E14.5. 24 hr after injection, embryos were isolated and embedded in O.C.T. media (Tissue-Tek) and stored at −80°C until sectioning using a cryostat (CM3050S; Leica). After sectioning, the Click-iTEdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dye (Invitrogen {"type":"entrez-nucleotide","attrs":{"text":"C10337","term_id":"1535408","term_text":"C10337"}}C10337), was used according to manufacturer’s instructions to evaluate EdU incorporation. Tissue sections were then co-stained for K10 (see 'Mouse tissue isolation, histology, and immunofluorescence staining'). Before imaging, coverglass was mounted onto slides using Prolong Gold with DAPI (Invitrogen {"type":"entrez-protein","attrs":{"text":"P36935","term_id":"549826","term_text":"P36935"}}P36935) and sealed with clear nail polish. Images were acquired with the Zeiss Imager M1 using Zen software. Total EdU-positive cells were counted and normalized to the total number of epidermal cells (determined by DAPI staining). Location of proliferating cells was determined by K10 staining (basal keratinocytes [EdU+/K10-], suprabasal keratinocytes [EdU+/K10+]) and then normalized to total EdU-positive cells. Statistical analysis was performed using Prism 8 software.