Mouse anti human oct3 4

Briefly, hPSCs were collected, washed with PBS, and incubated with 0.25% trypsin/EDTA for 8–10 min at 37°C followed by pipetting to dissociate. Trypsin activity was quenched by adding 2X volume of 20% FBS in RPMI supplemented with 5 μM Y-27632. Samples were centrifuged at 200g for 5 min and pelleted samples were fixed with 1% paraformaldehyde for 20 min at room temperature, permeabilized with ice-cold 90% methanol for 15 min at 4°C, and stored at −20°C until processing. Samples were washed twice with Flow Buffer 1 (0.5% BSA in PBS) to remove residual methanol, incubated for 1 hr at room temperature with primary antibodies in Flow Buffer 2 (0.5% BSA + 0.1% Triton X-100 in PBS), washed with Flow Buffer 2, and incubated in the dark for 30 min at room temperature with secondary antibodies in Flow Buffer 2. Samples were washed twice with Flow Buffer 2, resuspended in Flow Buffer 1, and stored on ice prior to analysis. Data were collected on a MACSQUANT flow cytometer (Miltenyi Biotec) and analyzed using FlowJo software. Primary antibodies and dilutions used were mouse anti-human Oct3/4 (Santa Cruz Biotechnology, sc-5279, 1:400), rabbit anti-human Nanog (Cell Signaling Technology, 4903S, 1:200). Secondary antibodies and dilutions used were goat anti-mouse AlexaFluor 488 and goat anti-rabbit AlexaFluor 647 (Thermo Fisher Scientific, 1:1000 each).

Samples were fixed with 10% neutral buffered formalin for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 5 min, blocked with 1% BSA in PBS for 30 min, and stained with primary antibodies (dilutions made in 1% BSA in PBS) for 1 hr at room temperature. Samples were washed three times with 0.05% Tween-20 in PBS and stained with secondary antibodies (dilutions made in PBS) for 1 hr at room temperature or overnight at 4°C. Nuclei were counterstained with DAPI. Primary antibodies and dilutions used: mouse anti-human Oct3/4 (Santa Cruz Biotechnology, sc-5279, 1:100), rabbit anti-human Nanog (Cell Signaling Technology, 4903S, 1:100), mouse IgG2a anti-βIII tubulin (R&D, MAB1195, 1:200), mouse IgG2b anti-alpha fetoprotein (R&D, MAB1369, 1:200), rabbit anti-alpha smooth muscle actin (Abcam, ab124964, 1:200), mouse IgG1 anti-PECAM-1 (EMD Millipore, MAB2184, 1:50). Secondary antibodies and dilutions used were goat anti-mouse AlexaFluor 488, goat anti-mouse IgG2a AlexaFluor 488, goat anti-rabbit or anti-mouse AlexaFluor 568, and goat anti-mouse IgG2b AlexaFluor 647 (Thermo Fisher Scientific, 1:1000 for all secondaries).