Immunohistochemical (IHC) and immunofluorecence (IF) staining
Primary antibodies that were used for IHC and IF are listed in Table S2.
Heat- induced antigen retrieval was performed with Antigen Unmasking
Solution, Tris-Based (Vector, H-3301) for all antibodies. For IHC
staining, endogenous peroxidase activity was blocked in 0.3%
H2O2 in deionised water. Nonspecific
binding was reduced by incubation in 2.5% goat serum. Sections were
treated with mouse Ig blocking reagent (Vector, MKB-2213-1) before
application of the primary mouse antibody. Rabbit primary antibody
binding was detected with goat anti-rabbit ImmPRESS HRP goat anti-rabbit
IgG (Vector, MP-741) and mouse primary antibody binding was detected
with Mouse-on-Mouse ImmPRESS anti-mouse Ig reagent (Vector, MP-2400).
Colour was developed with DAB-substrate chromogen system (Vector,
SK-4100). Images were acquired with a DM2500 Leica microscope (Leica
Microsystems).
For IF staining of aortic sections, rabbit primary antibody binding was
detected with goat anti-rabbit IgG (Alexa Fluor-488), mouse primary
antibody binding was detected with goat anti-mouse IgG (Alexa
Fluor-647), and goat primary antibody was detected with donkey anti-goat
IgG (Alexa Fluor-594). DAPI was used for nuclei visualization. Images
were acquired using an Olympus FV1000 confocal laser scanning microscope
with images analysed using Fiji54.
iVSMCs or HUVECs were grown on µ-Slide 8 well chamber slides (Thistle
Scientific) and fixed in 4% PFA. SVEP1, Integrin α4 and α9 staining was
performed on non-permeabilised cells. For all other staining, cells were
permeabilised in 0.5% Triton-X. Non-specific binding was reduced by
incubation in 1% bovine serum albumin (BSA), 22.5 mg ml-1 glycine,
0.1% tween-20 PBS solution, with additional blocking in a 10% goat
serum PBS solution. Cells were incubated overnight at 4°C in primary
antibody (listed in Table S2) diluted in 10% goat serum. After washing,
cells were incubated in 10% goat serum containing complementary
secondary antibodies. Nuclei were visualised by DAPI counterstaining.
Images were acquired using an Olympus FV1000 confocal laser scanning
microscope with images analysed using Fiji.