Axiocam 506 mono digital camera

Embryos were fixed in 4% PFA in PBS and infiltrated with 30% sucrose in PBS before being mounted in O.C.T. compound (Sakura Finetek USA Inc., Torrance, CA, USA). Sections (8 µm) were deposited on glass slides. Apoptotic cells were identified using the In Situ Cell Death Detection Kit, Fluorescein (Sigma-Aldrich Corp.) according to the manufacturer's instructions for the treatment of cryopreserved tissue sections. Sections were mounted in VECTASHIELDÒ Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and photographed using an Axiocam 506 mono digital camera (Carl Zeiss, Inc.) fitted onto an Axio Observed 7 fluorescence microscope (Carl Zeiss, Inc.). All positive signals were confirmed by DAPI staining. The percentage of TUNEL-positive cells was determined in three embryos per genotype per timepoint. Statistical analyses were performed with Prism 8 (GraphPad Software, Inc.) using a two-tailed, unpaired t-test with Welch's correction and Welch and Brown-Forsythe ANOVA tests.

Sections (8 µm) of PFA-fixed, sucrose-infiltrated, O.C.T-mounted embryos were deposited on glass slides. Sections were fixed in 4% PFA in PBS with 0.1% Triton X-100 for 10 min and washed in PBS with 0.1% Triton-X 100. Sections were blocked for 1 hr in 5% normal donkey serum (Jackson ImmunoResearch Inc., West Grove, PA, USA) in PBS and incubated overnight at 4°C in anti-Ki67 primary antibody (1:300; Invitrogen, Carlsbad, CA, USA) in 1% normal donkey serum in PBS. After washing in PBS, sections were incubated in Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody (1:1,000; Invitrogen) diluted in 1% normal donkey serum in PBS with 2 μg/mL DAPI (Sigma-Aldrich Corp.) for 1 hr. Sections were mounted in Aqua Poly/Mount mounting medium (Polysciences, Inc., Warrington, PA, USA) and photographed using an Axiocam 506 mono digital camera (Carl Zeiss, Inc.) fitted onto an Axio Observer 7 fluorescence microscope (Carl Zeiss, Inc.). All positive signals were confirmed by DAPI staining. The percentage of Ki67-positive cells was determined in three embryos per genotype per timepoint. Statistical analyses were performed with Prism 8 (GraphPad Software, Inc.) using a two-tailed, unpaired t-test with Welch's correction and Welch and Brown-Forsythe ANOVA tests.