Ds l1 camera

To visualize fast- and slow-ATPase activities of cardiac cells, two series of unfixed cryosections (5 μm thick) were treated. The assay is based on the different sensitivity of slow/fast ATPase to pH [27 (link),28 (link)]. Acid preincubation in 0.1 M sodium acetate buffer at pH 4.6 (10 min at 4 °C) in the presence of 10 mM ETDA inhibited the fast ATPase activity in a series of slides, while the other was incubated with 0.1 M sodium acetate buffer. All the slides were successively incubated in 0.5 mg/mL ATP in 0.1 M sodium acetate buffer at pH 9.4 (11 min at 37 °C), then treated with 2% COCl₂ for 5 min. Upon rinsing in distilled water, the sections were differentiated in ammonium sulfide for 30s to obtain a brownish-black precipitate of cobalt sulfide. Intensity of the color is associated to activity. The background signal in the absence of ATP in the reaction mixture was verified. (Supplementary Figure S2). Glycerin jelly-mounted sections were analyzed under Eclipse 55i microscope equipped with a DS-L1 camera (Nikon, Tokyo, Japan).

Frozen sections (5 μm thick) were air-dried, rinsed in phosphate-buffered saline (PBS) 0.05 M pH 7.4 for 5 min at room temperature, then transferred to 1 mM NADH/0.2 mM Nitroblue Tetrazolium/100 mM Tris-HCl buffer pH 8.0 containing 0.2% Triton X-100 solution for a maximum of 1 h at 37 °C to allow the oxidation of NADH and the formation of a stable blue formazan. The reaction was stopped by rinsing the sections in PBS. The sections were analyzed using an Eclipse 55i microscope equipped with a DS-L1 camera (Nikon, Tokyo, Japan) [23 (link),24 (link)].
The absence of an artifactual signal in the absence of the substrate in the reaction mixture has been verified (Supplementary Figure S1). Optical density of the blue formazan signal was assessed by FiJi software. At least 10 fields from 10× images were analyzed. In these fields the cardiomyocytes areas were selected as regions of interest where the average intensity of signal was measured and normalized versus the area of the region. Interstitium and arteries/arterioles were excluded from the measure.

Serial cryosections from the aortic root (10-micron thick) were obtained from 5 mice/group and were destined to either Hematoxylin/Eosin or Oil Red O or Sirius Red staining, following standard methods. Either lipids, collagen, or calcifications were measured in the aortic root cryosections. Images of aortic root sections were taken using the Eclipse55i microscope equipped with a DS-L1 camera and Lucia G software (all from Nikon, Tokyo, Japan). At least 6 sections/mice distanced 300 μm apart were obtained and analyzed by using ImageJ software. Either the total plaque areas, or the areas occupied by lipid, collagen and calcifications were selected as Region Of Interest (ROI). After conversion to binary, a suitable threshold was selected and the areas measured. For all the considered parameters, the percentage of areas/section was calculated and all the values/mouse/group were averaged. Reagents were purchased from BioOptica and Sigma.

Vascular sections were stained with hematoxylin/eosin and Movat’s pentachrome to assess morphological features. The presence of fibrous cap, foam cells, fibro-lipids, ulcerations, necro-thrombotic core, calcifications, hemorrhage/thrombosis and inflammatory infiltrates was determined in CPL [1 (link),51 (link)]. Sections were observed under an Eclipse55i microscope equipped with DS-L1 camera (all from Nikon, Tokyo, Japan) and composed to obtain whole section images. Samples showing evidence of blood stasis either due to intra-plaque hemorrhage or to surgical procedure were excluded from the study to avoid a potential bias in flow experiments.
Immunofluorescent labelling followed by confocal microscopy was performed as previously described [30 (link),32 (link)] to analyze vascular markers (Supplemental Table S3) Sections were examined under a Leica TCS-SP5-AOBS confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany); free projection max images were obtained from single channel acquired Z-series and merged by Adobe PhotoshopCS.