Biotinylated anti rabbit anti mouse igg

The rat testes tissue samples were fixed in 10% formalin, embedded in paraffin, and cut into 6 μm thin sections. The tissue sections were deparaffinized and then rehydrated with decreasing concentrations of ethyl alcohol (100%, 100%, 95%, 95%, 85% and 75%). Then, endogenous peroxidaseactivity was blocked by incubating the samples in 3% hydrogen peroxide followed byantigen retrieval. The specimens were then blocked with 5% bovine serum albumin (BSA) in room temperature for 30 min, followed by incubation with rabbit anti-CD34 (1:100 dilution; catalog no. BA3414; Boster, Wuhan, China), mouse anti-αSMA (1:100 dilution; catalog no. BM0002; Boster, Wuhan, China) antibodies at 4°C for 24 hours. Negative contral group was performed by 0.01 PBS (pH=7.4) at the same condition. After washing, the sections were incubated with biotinylated anti-rabbit/anti-mouse IgG (Boster Bio-Technology, Wuhan, China) for one hour at room temperature. The sections were then incubated with avidin-biotinylated peroxidase complex. Peroxidase activity was determined by staining with diaminobenzidine (DAB, Boster Bio-Technology, Wuhan, China). The nuclei were stained with hematoxylin.

The turtle pancreas samples used 10% formalin for tissue fixation. Tissue samples were first embedded and made into paraffin wax blocks, and then tissue sections were cut into 6 µm before being deparaffinized and rehydrated. Ethyl alcohol (100%, 100%, 95%, 95%, 85%, 75%) was then used in decreasing concentrations. Samples were incubated in 3% hydrogen peroxide to block endogenous peroxidase activity for retrieval of antigen. Bovine serum albumin (BSA) 5% was used for blocked specimens, which were placed for 30 min at room temperature, followed by incubation with rabbit anti-CD34 (1:100 dilutions; catalog no BA3414; Boster Wuhan, China) and mouse anti-αSMA (1:100 dilutions; catalog no. BM0002; Boster, Wuhan China) antibodies for 24 h at 4 °C. Sections were washed and incubated using biotinylated anti-rabbit/anti-mouse IgG (Boster Bio-Technology, Wuhan, China), then placed for 1 h at room temperature. The sections were incubated with avidin-biotinylated peroxidase complex. Next, the activity of peroxidase was determined by staining with diaminobenzidine (DAB, Boster Bio-Technology, Wuhan, China), and hematoxylin stain was used for the nucleus.