Alexa fluor 488 anti mouse cd45 antibody

Uterine horns of two adult littermate pairs were collected on 4.5 dpc after mating with a control male. The uterine horns were dissected and placed in ice cold RPMI media containing 1% HEPES, 1% Penicilin-Streptomycin, 1% L-glutamine and 0.5% Bovine Serum Albumin (BSA). Ovaries, oviduct and the cervix were removed and the remaining uterine horn was mechanically dissociated using blades. The minced tissue was placed in dissociation mix of the abovementioned media with collagenase type II, Dispase and DNAse I (Sigma) and incubated for 40 minutes at 37°C. After incubation, 0.5 mM EDTA was added and the tissue was further dissociated using a glass pipette. The remainder of the tissue was incubated for an additional 5 minutes at 37°C. The suspension was passed through a 70 μm filter and the flow through was centrifuged at 1,200 rpm for 5 minutes in an Eppendorf centrifuge (model 5702f). The cell pellet was resuspended and stained with Alexa Fluor 647 anti-mouse CD31 Antibody (# 102516, Biolegend), Alexa Fluor® 488 anti-mouse CD45 antibody (#103121, Biolegend) and DAPI for 20 minutes on ice. Endothelial cells (Cd31+, Cd45−, DAPI−) were sorted using a FACSJazz (BD Biosciences, San Jose, CA) cell sorter and RNA was immediately extracted using an Arcturus PicoPure RNA Isolation Kit (Applied Biosystems) according to the manufacturer’s instructions.