Ab6571

To evaluate the retention of collagen type I and IV, laminin and fibronectin, the decellularized and intact testes were fixed in 4% paraformaldehyde for 24 h followed by immersing in 30% sucrose as a cryoprotectant for 72 h; at the end, they were transported to the liquid nitrogen. The tissues were then embedded in OCT and sectioned at a thickness of 6–7 μm. Endogenous peroxidase was neutralized by 0.3% H₂O₂ in methanol, and non-specific binding sites were blocked by 4% goat serum in PBS. The samples were incubated with anti-collagen IV (pre-diluted; ab6581, Abcam), anti-collagen I (pre-diluted; ab6577, Abcam), anti-laminin (pre-diluted; ab6571, Abcam), and anti-fibronectin (pre-diluted; ab6584, Abcam) antibodies overnight at 4 °C. Then, they were washed with PBS three times for 15 min. Finally, the samples were treated with streptavidin–horseradish peroxidase complex followed by incubation with diaminobenzidine (DAB, Dako) and counterstained with hematoxylin.

Preservation assessment of collagen types I and IV, fibronectin, and laminin was performed by immunohistochemistry. Briefly, fresh unfixed frozen sections were prepared at 7 μm thickness. Quenching of endogenous peroxidase activity was done by 3% H₂O₂ diluted in methanol for 20 min. Nonspecific binding sites were blocked with 300 μL PBS containing 10% goat serum and 5% bovine serum albumin (BSA) for 1 hour at room temperature and, then, the sections were incubated with biotinylated anti-collagen type I (1:250; ab6577), anti-collagen type IV (1:500; ab6581), anti-fibronectin (1:250; ab6584) and anti-laminin (1:100; ab6571) antibodies overnight at 4 °C (All from Abcam PLC, Cambridge, MA, USA). After washing, 200–400 μL streptavidin-HRP (1:10000; Abcam, USA; ab7403) was added to each section and incubated for 20–30 min at room temperature. Eventually, DAB + chromogen-substrate system (1:50; Dako, Glostrup, Denmark; K3467) and hematoxylin counterstaining were used for detection. A matched similar protocol was done except using primary antibodies incubation as a technical negative control.