Immunofluorescence Staining of Aortic SMCs

Aortic SMC were fixed at 24 hrs after plating by immersion in 2% paraformaldehyde in Dulbecco’s phosphate buffered saline (DPBS). Cells were then washed in a glycine buffer and incubated overnight at 4 °C with primary antibodies against integrin α2 (Abcam, San Francisco, CA, USA), integrin α5 (Milipore Sigma, Burlington, MA, USA), and smooth muscle α-actin (SMα-actin) (Millipore Sigma, Burlington, MA, USA) diluted in a sodium citrate buffer containing BSA and Triton X [24 (link)]. After washing, cells were incubated with Alexa 568 secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature, washed again and immediately imaged in DPBS. A similar procedure with overnight incubation was followed for the primary antibody against integrin β1 or β3 both pre-conjugated with Alexa 488 (BioLegends, San Diego, CA, USA). For smooth muscle γ-actin (SMγ-actin, Actg2) staining, cells were first fixed with 1% paraformaldehyde in DPBS followed by permeabilization with cold methanol [25 (link)]. Staining was performed as described by using an SMγ-actin primary antibody [26 (link),27 (link)] followed by Alexa 488 secondary antibody (Jackson Immuno Research, West Grove, PA, USA).