Image pro plus v 5

Computerized image analysis of labeling was carried out using Image-Pro Plus v5.1 computer software (Media Cybernetics, USA) to quantify the percentage area of labeling for each of the molecules. We used an area of interest (AOI) tool to generate a series of areas that outlined the region in the outer laminae of Vc where Fos was expressed following pulpal stimulation (examples of these outlines are shown in Fig. 2). Outlines were made for each of the seven levels where Fos expression was quantified. These AOIs were superimposed onto captured images of pERK and pp38 labeling from equivalent rostro–caudal regions of Vc. The percentage area of the AOI that contained labeling for pERK and pp38 was calculated and an average of the seven levels used as the value for each animal.

For plaque component analysis, immunohistochemical and H&E staining was conducted as per our previous reports^{6 (link),48 (link)}. Briefly, aortic arch sections were incubated with primary antibodies for α-SMA (1:2,000 dilution, Servicebio, GB13044), Col3a1 (1:200 dilution, Abcam, ab6310), CD68 (1:100 dilution, ABclonal, A20803) and CD3 (1:100 dilution, ABclonal, A19017). After washing with PBS, the sections were incubated with secondary antibodies. For morphometric analysis, aortic arch sections were stained with H&E (Servicebio) and the collagen cap thickness was measured at the midpoint and shoulder regions of each lesion and quantified as the ratio of cap thickness to lesion size. Oil-Red-O staining was performed as previously reported^{6 (link),18 (link),50 (link)} and the entire aorta and 20-μm slices of aortic root were stained using Oil-Red-O (Sigma) to analyze the en face and cross-sectional atherosclerotic lesion areas, respectively. Images were analyzed by ImagePro Plus v.5.1 (Media Cybernetics).

PvNod22 10 μL aliquots of freshly purified protein (100 μg/mL in PBS pH 7.5) were applied to glow-discharged (15 mA/30 s) copper formvar/carbon-coated microscopy grids (200 mesh) and incubated at 20 °C for 10 min. After two washes with double-distilled water, samples were negatively stained with 2% (w/v) uranyl acetate. The stain was removed by washing with double-distilled water, and grids were air-dried and analyzed using a Carl Zeiss Libra (Oberkochen, Germany) 120 transmission electron microscope (TEM) operated at 120 kV. Electron micrographs were recorded at a nominal magnification of 60,000× and analyzed with Image-Pro Plus V5.1 (Media Cybernetics, Rockville, MD, USA).

The evaluation of the aortic intimal thickness was performed using image analysis [21 (link)]. Images were digitalized using a light microscope Eclipse 80i (Nikon Corp., Tokyo, Japan) with an attached digital camera (DS-2 MW, Nikon Corp.). Images were loaded to a computer equipped with the appropriate software (Image ProPlus v 5.1, MediaCybernetics, MD, USA) and the layers of the aorta (adventitia, media, intima) were measured. The thickness of the intima was calculated automatically.

ICAM‐1 (1:1000 dilution, ab206398; Abcam, Cambridge, UK), P‐selectin (1:1000 dilution, sc‐8419; Santa Cruz), E‐selectin (1:1000 dilution, DF6914; Affinity Biosciences, OH) antibodies were used in paraffin embedded heart sections. The results were analyzed by the multi‐function color cell image analysis system (Image‐Pro Plus V 5.1 software; Media Cybernetics company, Rockville, MD), and system automatically selects 10 meaningful vision, the integral optical density (IOD) was obtained for each view, and 10 views were chosen to calculated the mean IOD.