Dm500 microscope

Arterial images were acquired from NCs and patients with IPF by using a 4× objective in a vertical uni-direction to avoid overlap. We used a Leica DM 500 microscope with attached Leica IC50W digital camera for analysis. Using measuring tools from the ProPlus 7.0 program, the external length (from one end to the other end of the adventitia) for pulmonary arteries was measured. These measurements were used for arterial classification into six groups: 100–1000 µm (interspaced 100 or 200 µm), similar to the strategy used in our earlier study [3 (link)].

Immunohistochemical examinations were carried out based on the streptavidin-biotin immune-peroxidase technique. Four-μm paraffin sections were mounted, dewaxed, and rehydrated with phosphate buffered saline (PBS) (pH 7.2). Then, heat induced antigen retrieval was undertaken in citrate buffer (pH 6) for 20 minutes. In order to block the activity of endogenous peroxidase, sections were treated with 3% hydrogen peroxide for 10 minutes and washed in PBS. Overnight incubation of slides with the primary antibody anti TNF-α rabbit polyclonal antibody (1:50 dilution, Cat. No. A0277; AB clonal, Woburn, MA, USA) was undertaken. Then, slides were incubated with a secondary antibody and visualized by using the chromagen (3, 3’-diaminobenzidine tetrahydrochloride). Sections were counterstained with Mayer’s hematoxylin, washed with distilled water and PBS, dehydrated, and mounted. Light microscopic examination was done at the Department of Anatomy, Faculty of Medicine, Zagazig University. The images of the histological sections were obtained using a light microscope fitted with a digital camera (The Leica DM500 microscope, Leica ICC50 W Camera Modul, Cambridge, UK, Anatomy Department, Faculty of Medicine, Zagazig University, Egypt).