Tetramethyl rhodamine dutp

Inguinal fat fixed in 10% formalin was dehydrated through a graded ethanol series, embedded in paraffin, and cut into 5 μm sections. After staining with hematoxylin and eosin, sections were examined under a light microscope (OLYMPUS IX71) equipped with a SPOT RT color-2000 digital camera (Diagnostic Instruments, Sterling Heights, MI, USA). For uncoupling protein 1 (UCP1) immunostaining, sections were deparaffinized, rehydrated, and incubated with 0.5% Triton X-100, then blocked using 5% goat serum in phosphate-buffered saline (PBS). The primary antibody was rabbit antibody against human UCP1 (Abcam, Cambridge, UK), used at a dilution of 1:100 in PBS, whereas the secondary antibody was biotinylated goat anti-rabbit IgG antibody (Dako, Carpinteria, CA, USA), at a dilution of 1:250 in PBS. For detection of apoptosis, the TUNEL assay used terminal deoxynucleotidyl transferase and tetramethyl-rhodamine-dUTP (Roche Applied Science, Indianapolis, IN, USA) to directly label DNA strand breaks (red fluorescence). Images were acquired using a fluoromicroscope equipped with a SPOT RT color-2000 digital camera (Diagnostic Instruments, Sterling Heights, MI, USA).

Hydrated and permeabilized sections were treated for 1 h at 37°C with a solution containing 0.01 units/μl of terminal deoxynucleotidyl transferase enzyme (TdT) (Promega, Madison, WI, USA) in TdT buffer (pH 6.8) (Promega) and 3 nmol/ml of tetramethylrhodamine-dUTP (Roche Diagnostics, Mannheim, Germany). After incubation, sections were washed with PBS and counterstained with Hoechst 33342 (Sigma).