Immunocytochemistry Analysis of DR-ESCs

DR-ESCs were assessed for protein markers using immunocytochemistry and confocal microscopy. Cultures were fixed for 15 minutes at room temperature in 4% PFA, and immunostained as previously described^{37 (link),38 (link)}. Primary antibodies (Abs) used were: rat anti-mouse/human Oct3/4 at 1:200, rat anti-mouse platelet-endothelial cell adhesion molecule-1 (PECAM-1)/CD31 (BD Biosciences) at 1:1000, rabbit anti-mouse NG2 (Millipore) at 1:200, and rabbit polyclonal anti-phospho-Histone H3 (PH3, ser10, Millipore) at 1:500, rat anti-mouse PDGFRβ at 1:400, rabbit anti-mouse Desmin at 1:500, mouse anti-mouse A2B5 at 1:100. Secondary antibodies used were donkey anti-rat AlexaFluor647 (IgG; H+L) at ½ the primary Ab dilution factor (Invitrogen), and donkey anti-rabbit AlexaFluor647 (IgG; H+L) at ½ the primary Ab dilution. Incubation with DAPI for 30 min followed all staining. ESC cultures were imaged on a Zeiss LSM880 confocal microscope.

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Beth Payne L., Tewari B.P., Dunkenberger L., Bond S., Savelli A., Darden J., Zhao H., Willi C., Kanodia R., Gude R., Powell M.D., Oestreich K.J., Sontheimer H., Dal-Pra S, & Chappell J.C. (2022). Pericyte Progenitor Coupling to the Emerging Endothelium during Vasculogenesis via Connexin43. Arteriosclerosis, thrombosis, and vascular biology, 42(4), e96-e114.

Publication 2022

PECAM-1)/CD31 rabbit anti-mouse NG2 LSM880 confocal microscope

Antibodies Confocal microscope Dapi Desmin Dilution Donkey Escs Histone h3 Human Immunocytochemistry Mouse Mouse platelet endothelial cell adhesion molecule 1 Oct3 Pdgfrβ Protein Rabbit

Corresponding Organization : Carilion Roanoke Memorial Hospital

Other organizations : University of Virginia, Carilion Clinic, The Ohio State University, Duke Medical Center, Duke University Hospital, Duke University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein markers of DR-ESCs, including Oct3/4, PECAM-1/CD31, NG2, phospho-Histone H3 (PH3), PDGFRβ, Desmin, A2B5

control variables

Methods for fixing and immunostaining the DR-ESC cultures, including 15 minutes fixation in 4% PFA at room temperature, and staining protocol as described in references 37 and 38

controls

No positive or negative controls were explicitly mentioned in the protocol.

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