Anti laminin

Hearts were fixed with 4% formalin, embedded in paraffin, cut into 4 µm thick longitudinal sections and stained with hematoxylin-eosin. 10 µm frozen skeletal muscle sections of the lower leg of the hind limb were used to stain myofibril membranes using anti-laminin (Abcam) followed by anti−rabbit-Alexa 647 (Invitrogen) and capillaries with biotinylated isolectin B4 (Vector) followed by SAV-Alexa 488 (Invitrogen).

Proteins were extracted from mouse brain tissue using radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were separated on 8–10% SDS gels and transferred to polyvinylidene difluoride (PVDF) membranes. Western blot images were generated by cutting out individual portions of the whole membrane and incubating them with the respective antibodies after excision. The membrane was blocked with 5% skim milk at room temperature for 1 h, then anti-ZO-1 (1:200, Lifespan Biosciences, WA, USA), anti-laminin (1:1000, Abcam, Cambridge, UK), PECAM-1 (1:1000, LSBio, LifeSpan BioSciences, Seattle, WA, USA) and β-actin (1:10,000, Santa Cruz, CA, USA) were incubated overnight at 4 °C. The membrane was washed with PBST, and membranes were incubated with a 1:2000 dilution of horseradish peroxidase-conjugated anti-rabbit and mouse secondary antibodies for 2 h. Band detection was performed using chemiluminescence (Clarity™ Western ECL Substrate, Bio-Rad, Hercules, CA, USA) on the membrane. Analysis was performed using ImageJ to compare relative protein expression.

Monocultures of HUVECs and MSC-HUVEC co-cultures were grown on glass coverslips for 48 h or on Matrigel^TM for 19 h. HUVECs were pre-loaded with 5 μM CellTracker ™ Green CMFDA fluorescent dye (ThermoFisher Scientific, Waltham, MA, USA) for 40 min according to the manufacturer’s protocol. Cells were fixed with 4% formaldehyde and probed with the following primary antibodies (Abs): anti-CD31 (Biolegend, San Diego, CA, USA), anti-laminin (Abcam), anti-collagen 1 (Abcam), anti-collagen 4 (Abcam, Cambridge, UK), anti-fibronectin (Abcam, Cambridge, UK), or isotype-matched control immunoglobulins, followed by fluorophore-conjugated secondary Abs: goat anti-rabbit Alexa Fluor 594 (Invitrogen) or goat anti-mouse Alexa Fluor 594 (Invitrogen). Stained cells were visualized using a wide-field fluorescent Axiovert 200 M Microscope (Zeiss, Oberkochen, Germany).