Rabbit anti eomes

Embryos were fixed, stained, imaged, and recovered for genotyping as previously described [68] (link). Primary and secondary antibody sources were described previously [3] (link), [50] (link), and also included rabbit anti-EOMES (Abcam), rabbit anti-DAB2 (Santa Cruz Biotech), rabbit anti-LAMA1 (Sigma), and rat anti-PECAM1 (BD Biosciences).

Freshly sorted NK cells were seeded in a 96-Well Optical-Bottom Plate pre-coated with 100 μg/mL poly-L-lysin for 1 h incubation at 37 °C. After 4% PFA fixation for 15 min and three PBS washes, blocking step (PBS 3% BSA) for 30 min at RT was followed. Immunostainings were performed after a permeabilization step with 0.05% Triton X-100 for 7 min. Primary antibody antibodies were diluted in 3% BSA-PBS and added to the cell for one-hour incubation at room temperature. Primary antibodies used in this study include rat anti-CD122 (Bio X Cell, 4 μg/mL); rabbit anti-EOMES (Abcam, 4 μg/mL); FITC-conjugated mouse anti-T-BET (Biolegend, 1/50). After three washes with PBS, cells were incubated with the appropriate Alexa 555 conjugated anti-rat, AF647 conjugated anti-rabbit secondary antibody at 2 μg/mL in 3% BSA-PBS and add to the cells along with DAPI (4 μg/mL) for one-hour incubation at room temperature. After three gentle washes with PBS, cells were observed with a Zeiss LSM 800 laser scanning confocal microscope. The images and relative quantification were processed using Image J software.