Dab substrate kit

Testes section slides were deparaffinized with xylene and rehydrated in graded ethanol before being washed with distilled water for 5 min twice. The sections were treated with 3 % H₂O₂ to block endogenous peroxidase activity. Then, they were heated in (0.01 M) citrate buffer at 120 °C for 20 min in a microwave oven to retrieve antigen. The sections were cooled and washed three times with 0.01 M PBS, pH 7.2. Furthermore, they are blocked with goat normal serum in 0.01 M PBS for 30 min at room temperature. The antiserum-treated sections were incubated overnight at 4 °C with polyclonal antibodies against CASP3 and CLDN1, diluted at 1:500 and 1:1000, respectively. The second antibody (biotinylated anti-rabbit IgG) was added for 30 min, and then washed with PBS. After that, the horseradish peroxidase was added for 30 min. The bindings of the antibodies were visualized using the liquid DAB Substrate Kit (using diaminobenzidine as a chromogen substrate, Sigma). Tissue sections were counterstained with hematoxylin, dehydrated, cleared, and mounted with coverslips. The specificity of the antibody was examined using 1 % normal goat serum without the primary antibody. In regard to CASP3, the average number of positive cells was calculated in 30 fields/slide in five animals using magnification power ×200. Then, the data obtained were statistically analyzed.

Following 120 h of treatment, cells were collected and resuspended in PBS containing 1% BSA. A total of 5.0×10⁴ cells were concentrated onto glass slides using Cytospin 3 (Shandon, Runcorn, UK) at 650 rpm, for 5 minutes. The air dried slides were stained with May-Grünwald and Giemsa. For myeloperoxidase (mPOX) staining, cells were left in the dark for at least 24 h and stained using a DAB substrate kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's protocol. Photomicrographs of the stained cells were acquired using a Nikon microscope (Eclipse E1000M) equipped with Nikon Digital Sight DS-Fi1 camera.

Fixed AT samples were dehydrated, cleared and paraffin embedded. TH immunoreactivity was determined by DAB immunohistochemistry using 3 μm sections from 3 different levels (100 μm apart) per sample. Briefly, sections were dewaxed and endogenous peroxidase blocked by incubating in 3% HO₂ in methanol. Sections were then blocked in 3% rabbit serum and incubated overnight at 4 °C with anti-TH antibody (1/200) diluted in PBS. Bound primary antibody was detected using a biotinylated anti-sheep IgG secondary antibody, ABC reagent (Vector) and DAB Substrate kit (Sigma-Aldrich) according to the manufacturers’ instructions. Finally, sections were counterstained with hematoxylin. Parenchymal TH-positive fibers were analyzed by live count command in Lucia Imaging for image analysis (version 4.82, Nikon Instruments, Florence, Italy). The area of TH-positive nerve fibers was measured using images randomly captured with a Nikon E600 Eclipse microscope with camera. Ten areas from interscapular BAT were analyzed in each section. In inguinal ScW, 10 random areas were analyzed in each section. Areas of pure white adipocytes (larger, unilocular) and of pure beige adipocytes (smaller, multilocular) were analyzed separately.