Ds qi2 monochrome digital microscope camera

The cells were seeded on an 8-well slide chamber (BD Falcon Bedford, Franklin Lakes, NJ, USA) at a concentration of 5 × 10⁴ cells/well. After 24 h, the cells were washed in DPBS and fixed in 4% paraformaldehyde for 15 min at RT. Subsequently, fixed cells were permeabilized with 0.5%Triton-X 100 in DPBS for 15 min at RT and then blocked with 0.5%Triton-X 100, 10% FBS in DPBS (blocking solution) for 1 h at room temperature. Then, cells were incubated ON at 4 °C in a humid atmosphere with the primary antibodies diluted in blocking solution (Table 1). Negative controls were carried out by primary antibody omission. After being rinsed in PBS (3 times 10 min each), the cells were incubated with fluorochrome-labeled secondary antisera diluted in DPBS 1 h at RT (see Table 1). After 3 washes (10 min each) in PBS, slides were counterstained by DAPI and ultimately mounted. Images were obtained using a Nikon digital camera (DS-Qi2 Monochrome Digital Microscope Camera), installed on a Nikon epifluorescence microscope Eclipse E600 and analyzed with digital image software NIS-Elements BR Ver5.30.00 (Nikon, Tokyo, Japan).

Portions of 1 cm long portions from the aorta, pre and post enzymatic digestion, were embedded in OCT and frozen in isopentane cooled in liquid nitrogen. Ten-micrometer-thick sections were cut with a Leica CM1950cryostat (Leica, Wetzlar, Germany), then left to adhere to a microscope slide and stained with hematoxylin and eosin (H&E) according to the standard procedure. Images were obtained using a Nikon digital camera (DS-Qi2 Monochrome Digital Microscope Camera) installed on a Nikon epifluorescence microscope Eclipse E600 and analyzed with NIS-Elements BR Ver5.30.00 digital image software (Nikon, Tokyo, Japan).