Immunofluorescence Characterization of Vascular Tissues

Vascular tissues were harvested, washed using PBS and fixed for 6–8 h at 4 °C in 4% paraformaldehyde. Then, tissues were dehydrated in 30% sucrose solution overnight at 4 °C, embedded in OCT and frozen at − 80 °C. Frozen tissues were sliced into 10-μm thick sections using a cryostat (Leica, CM1950). Immunofluorescence staining was performed as described [27 (link)]. The primary antibodies used in this assay were αSMA antibody (Sigma, F3777, 1:500), SM22 (Abcam, ab14106, 1:200), CNN1 (Abcam, ab46794, 1:200), SMMHC (Abcam, ab53219, 1:200), RFP antibody (Rockland, 600-401-379, 1:50) and Ki67 (Abcam, ab53219, 1:200). The Alexa Fluor-conjugated secondary antibodies used in this study were Donkey anti-Mouse IgG (Invitrogen, A21202 for Alexa Fluor 488), Donkey anti-Rabbit IgG (Invitrogen, A31572 for Alexa Fluor 555), Donkey anti-Rabbit IgG (Invitrogen, A32731 for Alexa Flour 488) and Donkey anti-goat IgG (Invitrogen, A32816 for Alexa Fluor 555). Images were taken using the Nikon A1 confocal microscope. The co-staining area was quantified using the colocalization tool of Fiji. The mean gray value was calculated using the Fiji measurement tool.