Tissue clearing using the clear unobstructed brain imaging cocktails and computational analysis (CUBIC) protocol and multi-photon deep imaging

GFP transgenic male mice were anesthetized with sodium pentobarbital (50 mg/kg). The mice were then fixed in a supine position with their necks extended and were then transcardially perfused through the left ventricle with heparin containing (10 U/mL) saline, followed by fixation with 4% paraformaldehyde in phosphate buffered saline (PBS). After perfusion, a median incision was made to open the abdominal cavity to harvest bilateral pars vas deferens. The dissected specimens were immersed in the same fixative overnight at 4 °C. After washing with PBS, the post-fixed specimens were immersed in 50% CUBIC-L solution (T3740, Tokyo Chemical Industry) in distilled water on a shaker at 37 °C overnight. Next, the specimens were immersed in 100% CUBIC-L solution on a shaker at 37 °C for 5 days and 100% CUBIC-L solution was refreshed every other day. Following delipidation, the specimens were washed in PBS overnight at room temperature (22–25 °C). The specimens were then immersed in 50% CUBIC-R⁺ solution (T3740, Tokyo Chemical Industry) in distilled water on a shaker at room temperature (22–25 °C) overnight, and then immersed in CUBIC-R⁺ solution on a shaker at room temperature for an additional 1 or 2 days and later embedded in CUBIC-R⁺. The transparent specimens were observed under a multi-photon microscope (FVMPE-RS, Olympus, Japan) with acquisition parameters as follows: Excitation at 1000 nm, a 10 × 0.6NA SCALEVIEW-A2 immersion lens (XLPLN10XSVMP, Olympus, Japan) and an image size of 1271 × 1272 μm. Image deconvolution and three-dimensional (3D) reconstruction were performed with the resulting image stacks and was analyzed using Avizo software (version 9.1.1, FEI, USA) available at https://www.fei.com/software/avizo3d/.