Ab21027

Cell cultures or pcgENS (at day 11) were fixed with 4% paraformaldehyde in PBS at room temperature for 30 min. After permeabilization with PBS-sodium azide containing 10% horse serum and 0.25% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA), samples were incubated at room temperature, sequentially, with primary antibodies for 3 h, washed 3 times with PBS, and then with secondary antibodies for 1 h, diluted in PBS-sodium azide containing 10% horse serum. The following primary antibodies and dilutions were used for immunofluorescence microscopy experiments: rabbit anti S100β (1:200, IR504, Dako, Santa Clara, CA, USA) staining glial cells; goat anti α-smooth muscle actin (SMA) (1:200, ab21027; Abcam, Cambridge, UK) staining smooth muscle; mouse anti PGP9.5 (1:500, MA1-83428, Thermo Fisher Scientific, Waltham, MA, USA); rabbit anti PGP9.5 (1:500, CL7755AP, Cedarlane, Burlington, ON, Canada) staining enteric nerves and rabbit anti-N-Cadherin (1:200, Ab-18203, Abcam). The following secondary antibodies were used: anti-mouse Cy3 (1/500, 715-165-151, Jackson ImmunoResearch, West Grove, PA, USA), anti-mouse FP 488 (1/500, 115-545-205, Jackson ImmunoResearch), anti-rabbit Cy3 (1/500, 711-165-152, Jackson ImmunoResearch), and anti-goat AMCA (1:50, 705-155-147, Jackson ImmunoResearch).

Excised vascular segments were dissected to exclude regions where ligations were placed or anastomoses. For samples from ApoE knockout mice, the segment between the ligations, as well as the zone of bifurcation was analyzed. Vascular segments were fixed in 4% paraformaldehyde, embedded in an upright position in paraffin, cut sequentially into 4μm sections and subjected to hematoxylin and eosin staining (H&E) or immunofluorescence staining. The distance between sections ranged from 200μm to 300μm. For immunofluorescence, the following antibodies were used at indicated concentrations: anti-ACTA2 (Abcam, ab21027) (1:1000), anti-ICAM-1 antibody [YN1/1.7.4] (Abcam, ab119871) (1:200). Secondary antibodies were Alexa Fluor 546 (Invitrogen, A11056) and Alexa Fluor 647 (Jackson Immuno-research, 712-606-153), both utilized at a 1:200 dilution.
Quantification of nuclear sizes was performed by ImageJ on H&E sections. IH and atherosclerosis were analyzed on H&E sections.

3 randomly selected, formalin-fixed tissues from Saline- and Au@Ag-treated groups were routinely embedded in paraffin, then 4 mm thick paraffin slices were cut. Samples were deparaffinised, then heat-mediated antigen retrieval was performed using citrate buffer at pH = 6. Samples were blocked with donkey serum (Millipore) and stained with alphaSMA-specific antibody (1:200) (abcam, ab21027) then Alexa488-labelled donkey-anti-goat secondary antibody (abcam, ab150129) was applied. After alphaSMA staining, samples were blocked again using normal goat serum, and Ki67-specific antibody (abcam, ab15580) was used in 1:50 dilution followed by Dylight549-labelled goat anti-rabbit secondary (abcam, ab96984). After immunoreactions, samples were counterstained with DAPI and analysed by an Olympus BX51 fluorescent microscope.
For correlation analysis, 4–5 photographs were taken from the subcutaneous regions of each tumour, then the percentage of alphaSMA- and Ki67-positive cells was quantified using CellProfiler 2.2.0 software. Statistical evaluation of the obtained data was performed using GraphPad Prism 7 software by calculating the Pearson r value.