1. The testicular tissues were immediately transported to the laboratory on ice and washed to remove any residual blood.
2. Testicular tissues were separated into single seminiferous tubules with sterile tweezers and cut into 1 cm sections for a total length of 10 cm. (10 min)
3. Single seminiferous tubules were exposed to a DMEM/F12-based vitrification solution containing 1.05 M DMSO and 1.35 M EG (E-9129; Sigma-Aldrich) for 10 min and then to a solution with 2.1 M DMSO and 2.7 M EG for 3 min. In the second step of vitrification, the vitrification solution was supplemented with 20% HSA and 0.5 M sucrose.(15 min-20 min)
4. For microinjection, vitrification solution was introduced into the seminiferous tubules via microinjection needles connected to a microinjection pump with a flow rate set at 2 μl/s. Under microscopy, the perfusion needle was inserted into one end of the inferior tubule for 2–3 sec. The volume of perfusion fluid injected was approximately 4–5 μl. The microinjection was performed in both vitrification steps described above
5. Then the microinjected tissue pieces were cut into 2 mm lengths and transferred to the frozen carrier “Cryopiece”(Sun et al., 2017) and immersed in liquid nitrogen to allow vitrification. (5 min)
6. Samples were thawed by adding a pre-warmed solution (37°C, DMEM/F12 + 1 M sucrose + 10% HSA) to the cryovials and kept at 37°C for 1 min. Then the samples were washed in DMEM/F12 with 10% HSA and 0.5 M sucrose for 3 min at 37°C, then washed in DMEM/F12 with 10% HSA for 5 min at 37°C. (15 min)