Horseradish peroxidase labeled streptavidin

The protein expression levels of Fas, Bcl-xl and leptin receptors were tested by immunohistochemistry. The sections were deparaffinized and were subsequently boiled for 15 min in sodium citrate buffer for antigen retrieval. Following elimination of internal peroxidase activity, the sections were incubated with rabbit anti-Fas (1:50; cat no. BA0408), rabbit anti-leptin-receptor (1:50; cat no. BA1233; Wuhan Boster Biological Technology, Ltd., Wuhan, China) and rabbit anti-Bcl-xl (1:100; cat. no. 10783-AP; Proteintech Group, Inc., Chicago, IL, USA) antibodies at 4°C overnight. The tissue sections were exposed to biotinylated sheep anti-rabbit immunoglobulin G solution (Proteintech Group, Inc.) at 37°C for 30 min and were subsequently incubated with horseradish peroxidase-labeled streptavidin (Proteintech Group, Inc.) at 37°C for 30 min. Finally, the tissue sections were observed under light microscopy (Nikon E100; Nikon), and the photomicrographs were obtained by Photo Imaging System (Canon 600D; Canon). In each group 30 fields (5 fields/rat) in 6 rat testis were randomly selected. Positive expression was assessed using Image-Pro Plus 6.0 and a mean of the integrated optical density was obtained.

Decidua tissue was collected and washed by PBS three times. The decidua tissues were then put into 10% formaldehyde at room temperature for 24 h, followed by deparaffinization and dehydration. Then, the samples were cut into 5-µm sections for further immunohistochemistry detection. Endogenous peroxidase was blocked with 3% H₂O₂. Non-specific staining was blocked using 5% bovine serum albumin for 20 min. Subsequently, the sections were incubated with primary antibody targeted to ADAMTS-7 (1:200) and COMP (1:200) at 4°C overnight. Thereafter, the decidua sections were exposed to biotinylated sheep anti-rabbit immunoglobulin G solution (Proteintech Group, Inc.) at 37°C for 30 min followed by incubation with horseradish peroxidase-labeled streptavidin (Proteintech Group, Inc.) at 37°C for 30 min. After washing with PBS three times, the sections were counterstained with hematoxylin and dehydrated. Then, the sections were observed under a microscope (Nikon E100; Nikon Corporation, Tokyo, Japan), and the photomicrographs were obtained by Photo Imaging System (Canon 600D; Canon, Inc., Tokyo, Japan). A brown color was regarded as a positive signal. The optic density was detected by Image-Pro Plus 6.0, and the mean of the integrated optical density was obtained to represent the expression of ADAMTS-7 and COMP.