B40 microscope

Lung tissues were sectioned at 4 μm thickness and stained with mouse monoclonal Ki-67 antibody (Dako, Glostrup, Denmark) diluted 1:200. The DAKO ARK™ (Animal Research Kit) (Dako, California, United States) detection was used according to the manufacturer's instructions. Briefly, the sections were deparaffinized and dehydrated with xylene and graded alcohols. Antigen retrieval was carried out by heating for 30 min at 110°C in Tris-EDTA solution pH 9, followed by peroxidase blocking to quench endogenous peroxidase. Sections were then incubated for 15 min with the prepared biotinylated primary antibody, followed by streptavidin-peroxidase incubation for 15 min. Finally, 3,3′Diaminobenzidine (DAB) substrate chromogen was applied onto the sections for 5 min and counterstained with Mayer's Hematoxylin solution.
Three random fields of each lung tissue section were image-captured using high magnification (400×) with a B×40 microscope (Olympus Corporation, Tokyo, Japan). Two hundred nuclei in each field were counted manually, and a total of 600 nuclei per tissue section were analyzed. An average percentage of positively stained cells for every 100 nuclei were calculated as the proliferation index.

Right ventricular specimens were fixed in 4% paraformaldehyde, washed in phosphate‐buffered saline (PBS), embedded in paraffin, and 4 µm sections were obtained. Immunohistochemistry was performed using the ABC‐Kit from Dakocytomation. Slides were observed with an Olympus B40 microscope. Pictures were taken using a Media Cybernetics camera (Bethesda, MD, USA) and pro‐image plus software (Bethesda).

Cell death was investigated with the “in situ cell death detection” kit from Roche. Rat neonatal cardiomyocytes were cultured in 4-well chamber slides (Nunc, UK) at a density of 1.5 × 10⁵ cells per cm². Cells were washed twice with PBS and permeabilized for 1 hour at room temperature with 0.1% Triton (v/v) in 0.1% sodium citrate (v/v). They were then washed three times in PBS and incubated with the labelled enzyme for 1 hour at 37°C. They were washed twice, mounted in VECTASHIELD containing DAPI, and observed with an Olympus B40 microscope. Apoptosis was quantified by counting apoptotic cells in 25 different fields or 400 cells. Pictures were taken using a Media Cybernetics camera (Bethesda, MD, USA) and analysed with proimage plus software (Bethesda, MD, USA).