Immunofluorescent staining, microscopy, and quantification

For MED13 staining, embryos were fixed in ice-cold methanol for 10 min, then blocked overnight at 4°C in blocking buffer (PBS, 3 mg/ml BSA, 0.01% Tween-20) supplemented with 10% fetal calf serum. Embryos were then incubated in primary anti-MED13 antibody (Santa Cruz, Dallas, TX, sc-5369; 10 μg/ml) for 1 h at room temperature (RT) followed by Alexa Fluor 488 anti-goat IgG (Thermo Fisher Scientific, cat# A-11055; 2 μg/ml) for 1 h at RT. For γH2AX staining, embryos were fixed in 4% paraformaldehyde for 30 min, permeabilized in 0.1% Triton X-100 for 20 min, then incubated in blocking buffer for 1 h. Embryos were incubated in primary γH2AX antibody (Bioworld Technology, St Louis Park, MN; BS4760; 5 μg/ml) at 4°C overnight followed by Alexa Fluor 555 anti-rabbit antibody (Thermo Fisher Scientific, cat# A-21429; 2 μg/ml) for 1 h at RT. For MED13L staining, embryos were fixed in 2% paraformaldehyde for 40 min, permeabilized as above, then washed in blocking buffer. Embryos were incubated in primary MED13L antibody (Bethyl Laboratories, Montgomery, TX, A302-421A; 10 μg/ml) at 4°C overnight followed by Alexa Fluor 555 anti-rabbit antibody (Thermo Fisher Scientific, cat# A-21429; 4 μg/ml) for 1 h at RT. For NANOG staining, embryos were fixed in 4% paraformaldehyde for 30 min, permeabilized in 0.1% Triton X-100 for 20 min, then incubated in blocking buffer for 1 h. Embryos were incubated in primary NANOG antibody (R&D Systems, Minneapolis, MN, cat# AF2729; 10 μg/ml) at 4°C overnight followed by Alexa Fluor 647 anti-goat IgG (Thermo Fisher Scientific, cat# A-21447; 4 μg/ml) for 1 h at RT. Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) was performed as described previously [29] using the In Situ Cell Death Detection Kit, Fluorescein (Sigma-Aldrich) except that 1.5 μg/mL DAPI was in the mounting medium. Peroxide-treated embryos served as positive controls [29]. For some experiments, zona pellucidae were removed immediately prior to fixation using acid Tyrode medium.

For cell counts, blastocysts were fixed 120 h after hCG injection in 2% paraformaldehyde for 40 min, permeabilized as above, then washed in blocking buffer. Embryos were incubated in primary anti-CDX2 antibody (Abcam, Cambridge, MA; ab157524; 1 μg/ml) at 4°C overnight followed by primary OCT-3/4 antibody (Santa Cruz, Dallas, TX; sc-8628; 10 μg/ml) for 1 h at RT. The embryos were then washed and incubated with Alexa Fluor 488 donkey anti-mouse (Thermo Fisher Scientific, cat# A-21202; 4 μg/ml) and Alexa Fluor 555 donkey anti-goat (Thermo Fisher Scientific, cat# A-21432; 4 μg/ml) for 1 h at RT.

All oocytes and embryos were mounted in Vectashield containing 1.5 μg/mL DAPI (Vector Laboratories, Burlingame, CA), and slides were scanned using a Zeiss LSM 510 UV confocal microscope. Immunofluorescence signals were quantified using ImageJ software. For EU, EdU, and γH2AX staining, a region of interest was drawn around each nucleus and the average pixel intensity was determined. The values for each nucleus were then averaged to generate an intensity level for each embryo. Intensity was expressed relative to the average intensity of the control embryos for each independent experiment. Quantification of CDX2, OCT4, and NANOG staining intensity was done from confocal z-stack scans through entire blastocysts followed by automated measurement of signal intensity in each slice using ImageJ. The total image intensity was calculated by multiplying the overall average intensity by the number of images in the stack. Quantification of blastocyst inner cell mass (OCT4-positive), trophectoderm (CDX2-positive), and total cells (DAPI) was performed manually after scanning a z-stack of images through the full depth of each embryo. Total cell numbers were counted after encoding nuclear depth using a rainbow look-up table as described previously [30].