Quantifying LINE-1 ORF1p in Blastocysts

Confocal analyses of LINE-1 ORF1p expression were analysed on a Zeiss LSM 710 confocal microscope using a previously described method [91 (link)]. Antibodies for the immunostaining: Rabbit anti LINE-1 ORF1p, 1:500, a generous gift of Dr. Oliver Weichenrieder (Max Planck, Germany). Secondary antibody: Alexa 488 Donkey anti Rabbit, 1:1,000 (Thermo). Mouse anti γ^+-H2AX, 1:200, clone 3F2 (Novus). Secondary antibody: Alexa 555 Donkey anti Mouse, 1:1,000 (Thermo). Rabbit anti cleaved caspase-3 (cl_Caspase3) (Cell Signalling, 9661S) was used at a 1:200 dilution. Secondary antibody: Alexa Fluor anti Rabbit 555 (1:1,000, Invitrogen). DAPI (Thermo) was used at 1:500. To assess the number of L1-ORF1p cytoplasmic foci in human blastocysts, every second image of a confocal stack was used at the height of the ICM. L1-ORF1p cytoplasmic foci were enhanced by applying a granulation filter with the same parameters to all images, and ORF1p cytoplasmic foci were counted manually. To assess L1-ORF1p⁺- cl_Caspase3⁺ double staining, 6 embryos were stained. To quantify the L1-ORF1p⁺-Cl-csapase3⁺ cells, 4/6 embryos were used.