In situ cell death detection pod kit

Left lungs were fixed by perfusion with 0.1 M phosphate buffered saline (PBS; pH 7.4) containing 4% paraformaldehyde (PFA). Trachea was ligated with a surgical suture, and lungs were incubated in fresh 4% PFA-PBS solution on ice for 4–5 h. The lungs were paraffin-embedded for obtaining 5 μm sections which were then mounted onto poly-L-lysine-coated slides (Paul Marienfeld GmbH&Co., Lauda-Konigshofen, Germany), stained with standard haematoxylin-eosin and Masson’s trichrome techniques for histopathologic evaluations and with ABC technique for lamellar body membrane protein (LBMP) expression as described previously [15 (link)–17 (link)].
Apoptosis was evaluated by Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) technique using in situ cell death detection POD kit (Roche Molecular Biochemicals, Mannheim, Germany) as described previously [15 (link)].

Rats on the four experimental groups were sacrificed 6 days postpartum (n = 4 per experimental group). Animals were decapitated. After enucleating, anterior segments of the eyes, including the lens, were discarded, and the posterior segments of the eyes containing the retinas were fixed in 4% paraformaldehyde in 0.1 M pH 7.4 phosphate buffer at 4°C for 48 h. Tissues were dehydrated and paraffin-embedded. Tissue sections (5 μm-thick) were stained for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) with the in situ Cell Death Detection POD Kit (Roche, Basel, Switzerland), following manufacturer’s instructions. Visualization of immunoreactivity was performed with 0.03% 3,3′diaminobenzidine (Sigma Co, St. Louis, MO, USA), 3% nickel ammonium sulphate and 0.01% hydrogen peroxide diluted in 0.1 M buffer acetate, yielding a black product.
The animals used for electroretinography were intraperitoneally anesthetized with ketamine/xylazine and intracardially perfused with the same fixative. The posterior segments of the eyes were paraffin-embedded, sectioned, and stained with hematoxylin-eosin to count the number of ganglion cells and to measure the thickness of the most inner layers of the retina (IR), which includes the internal limiting membrane, the retinal optic nerve fiber layer, and the ganglion cell layer (GCL), as reported (Rey-Funes et al., 2013 (link)).

The prostate tissues in each group were dewaxed with xylenes and rehydrated through an ethanol series. Tunnel reactions were performed with the In Situ Cell Death Detection POD Kit (Roche USA) according to the manufacturer's protocol. The prostate tissues after the Tunnel reactions were dehydrated, mounted, and examined under a microscope.