E7889

VSMC cultures were trypsinized at cell density of 200,000 cells/5 mL in T25 flasks and then fixed with 4% paraformaldehyde during 10 min at room temperature. VSMCs were permeabilized with 0.5% saponin (S7900, Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS) for 5 min and blocked with 1% BSA (A6003, Sigma-Aldrich, St. Louis, MO, USA) for 30 min. Primary antibodies used were anti-alpha smooth muscle actin at 1/100 (ab66133, Abcam, Cambridge, UK), transgelin/SM22 monoclonal antibody at 1/100 (60213-1-Ig, ProteinTech, Manchester, UK) and anti-CD31 (also called platelet and endothelial cell adhesion molecule 1, PECAM1) [EPR3094] antibody at 1/50 (ab76533, Abcam, Cambridge, UK) during 1 h in shaking at room temperature. VSMCs were washed with 5 mM EDTA (E7889, Sigma-Aldrich, St. Louis, MO, USA) and 0.5% BSA in PBS and were incubated with secondary antibodies Alexa Fluor^® 488 goat anti-mouse IgG (H + L) (A-11001, Invitrogen, Thermofisher, Carlsbad, CA, USA) and Alex Fluor^® 647 goat anti-rabbit IgG (H + L) (A-21244, Invitrogen, Thermofisher, Carlsbad, CA, USA) at 1/500 for 30 min in shaking and darkness. After washing, cells were resuspended in 5 mM EDTA and 0.5% BSA in PBS.

Mice were deeply anesthetized by injecting 100 μg pentobarbital intraperitoneally. Blood was collected from the right ventricle prior to perfusion. Briefly, 200 μL blood were collected into tubes containing ethylenediaminetetraacetic acid (EDTA, E7889, Sigma, Sweden), lysed in ACK buffer (A1049201, Gibco) and then centrifuged. The pellet was resuspended in cold PBS and used for staining. Spleens were dissected and cell suspensions prepared by mechanical dissociation in ice-cold PBS by passing through 40 μm cell strainers (734-0002, VWR; Stockholm, Sweden). Mice were perfused through the left cardiac ventricle using ice-cold PBS. Whole brain and spinal cord were removed and minced with a surgical disposable scalpel (Medicarrier AB, Sweden), followed by enzymatic digestion using Collagenase (11088866001, Roche, Sweden) and DNAse (000000010104159001, Roche, Sweden). Myelin was removed using 38% Percoll (P1644-1L, Sigma, Sweden).