Proimage plus software

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).

Right ventricular specimens were fixed in 4% paraformaldehyde, washed in PBS, and 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 analysed with proimage plus software (Bethesda, MD, USA).