Ksom hepes buffer

Wild-type 129/SvEv mice were euthanized 3.5 days post-mating. The uterus was isolated and washed in KSOM-HEPES buffer (Milli-pore Sigma). The utero-tubule junctions were cut and the uterine horns were flushed using a 25 gauge needle filled with 0.4 mL of M2 medium (Millipore Sigma). Using a mouth controlled Pasteur pipette, blastocysts were collected and transferred into a drop of PBS. Blastocysts were hatched by serial passage through drops of Acid Tyrodes (Millipore Sigma). Hatched blastocysts were transferred into drops of PBS, and then fixed in 3% paraformaldehyde and VVL immunofluorescence staining was performed as described under Vicia villosa lectin assays. Blastocyst images were obtained using EVOS epifluorescence and Nikon A1 laser scanning confocal microscope. Immunostaining of the trophectoderm of intact, infected blastocysts was performed 48 hours post-infection. The shControl or shGalnt3 infected blastocysts were fixed with 3% paraformaldehyde and blocked for 30 min using 1X carbofree block. Blastocysts were stained for 1 hour using VVL-FITC (Vector Laboratories) at room temperature. The VVL-FITC stained blastocysts were imaged at 20X using EVOS microscopy.