Image pro plus v 6.0 image

The kidneys were longitudinally sliced from the middle into two and immersed in 10% paraformaldehyde. The renal tissues were embedded in paraffin wax and 5 mm slices were deparaffinized and rehydrated. After the microwave antigen retrieval (citrate buffer, pH 6.0) was used for 10 min, slices were dipped in 3% hydrogen peroxide to block endogenous peroxidase for 10 min. After that, these were treated with 10% normal goat serum (diluted in Tris-buffered saline) at 37 C for blocking nonspecific binding sites. Subsequently, incubated the slices overnight at 4 C with rabbit anti-rat RGC32 (1:200, Santa Cruz Biotechnology, Dallas, TX). After washed with phosphate-buffered saline (PBS), these slides were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody for 30 min at room temperature. 3,3-diaminobenzidine tetrahydrochloride (DAB) was used to for developing color. Counterstaining was performed with hematoxylin. Ten areas (Â400) were acquired randomly with microscopy imaging system (Leica Microsystems, Wetzlar, Germany) and were analyzed by the Image-Pro Plus v 6.0 image analysis software (Media Cybernetics, Bethesda, MD). The mean optical density of the images (Â400) was designated as representative RGC32 staining intensity. The researcher had no knowledge of the group assignment.