3 3 diaminobenzidine dab solution

The 8-mm paraffin sections cut on microscopy slides (Muto pure chemicals co., LTD, Tokyo, Japan) were deparaffinzied and rehydrated using a decreasing ethyl alcohol gradient followed by phosphate buffer saline (PBS). The sections were then blocked with normal goat serum (Fisher scientific, Hampton, VA, USA). The sections were incubated with a 1:200 dilution of primary antibody overnight at 4 °C and washed with PBS. The slides were incubated with a 1:200 dilution of secondary antibody for 30 min at 24 ± 3 °C. After washing thrice in PBS, the slides were incubated with 1 drop (100–200 μL) 3,3′-diaminobenzidine (DAB) solution (abcam^®, Cambridge, MA, USA) for 1–2 min at 24 ± 3 °C. Finally, the slides were counterstained using hematoxylin solution. Images were acquired using a Leica DM IL LED microscope (Leica^®, Wetzlar, Germany).

Prostate tissues from the rats were fixed in 10% buffered formalin and embedded in paraffin. The tissue blocks were cut into 8 μm thickness and stained with H&E prior to histological evaluation. Images of the epithelial thickness were acquired using the Leica Application Suite software (LAS version 3.3.0, Leica Microsystems, Inc., Wetzlar, Germany). The tissue sections were placed onto microscopy slides (Muto Pure Chemicals Co., LTD., Tokyo, Japan), deparaffinized, and the endogenous peroxidase activity was depleted. The sections were then blocked with normal goat serum and incubated overnight at 4 °C with a primary antibody (1:200). The slides were then incubated with corresponding horseradish peroxidase-conjugated secondary antibody (1:500) and incubated at room temperature for 2 h. After washing thrice in PBS, the sections were incubated with 3,3′-diaminobenzidine (DAB) solution (Abcam, Cambridge, MA, USA). Finally, the tissues were counterstained with hematoxylin and mounted with mounting medium (Agilent Technologies, Inc., Santa Clara, CA, USA). Images were acquired using NIS-Elements F (version 4.0., Nikon, Tokyo, Japan).