Nb120 6586

For cell imaging after 7 days of culture to visualize VM structures collagen gels were fixed using two washes of 4% PFA for 30 min each at room temperature. F-actin was stained using AlexaFluor® 488 Phalloidin (Cell signaling technology, Danver, MA) and the nuclei were counterstained with DAPI. For immunofluorescence staining the gels were incubated with the primary antibody for 48–72 h. Anti-COL4A1 (1:200 dilution, NB120-6586, Novus Biologicals).

For western immunoblot analysis, conditioned perfusion medium was collected. Concentrating beads (StrataClean Resin, Agilent) were added to the conditioned medium and centrifuged, after which the supernatant was discarded. An equal volume of 2X Laemmli buffer was added, boiled, and separated by SDS-PAGE. Protein was transferred to nitrocellulose membranes and blocked with SuperBlock T20 (Thermo Fisher Scientific). Blots for the conditioned medium were incubated with one of the following primary antibodies overnight at room temperature: rabbit anti-COLIV (1:1000, NB120-6586; Novus), and mouse anti-FN (0.5–1 ug/ml, AB200541; Abcam). The blots were then incubated for 1 h at room temperature with HRP-linked anti-mouse or anti-rabbit secondary antibodies (goat anti-rabbit, SC-2004, 1:1,000, goat anti-mouse, SC-2005, 1:1,000). Chemiluminescent signal was developed with SuperSignal West Femto Substrate (Thermo Fisher Scientific) and images were captured with the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA). Original blots depicted in Fig. 4 have been displayed as uncropped blots in Supplementary Fig. 2. All blots derive from the same experiment and were processed in parallel.

Immunohistochemistry (IHC) staining was used to evaluate COL4A1 protein expression. The COL4A1 antibody (NB120-6586) was purchased from Novus Biologicals (Novus Biologicals, Littleton, CO, USA). Please also have a look at NB120-6586">https://www.novusbio.com/products/collagen-iv-alpha-1-antibody_NB120-6586#PublicationSection. The COL4A1 antibody (NB120-6586) has been tested in human, mouse, rat, and bovine and applied to immunocytochemistry (ICC), immunofluorescence (IF), immunohistochemistry (IHC-frozen sections and paraffin-embedded sections), enzyme-linked immunosorbent assay (ELISA), and western blot (WB). Previously described IHC evaluation and protocol were used to obtain score [13 (link)]. The mean signal scores were evaluated independently by the two pathologists who were blinded when assessing the samples. Immunostaining scores were defined as the cell staining intensity (0 = nil; 1 = weak; 2 = moderate; and 3 = strong) multiplied by the percentage of labeled cells (0% to 100%), leading to scores from 0 to 300. The mean of score of signals was evaluated independently by the two pathologists. The median IHC staining score (75) was used as the cutoff point for the dichotomization of COL4A1. A score greater than 75 was defined as “high” immunostaining, whereas a score of less than or equal to 75 was defined as “low.”