Leica rm2235

Teratoma analysis was performed as described previously [72 (link)]. Briefly: iPSCs were trypsinized, washed in PBS, and 10⁶ cells were subcutaneously injected in the hindlimb of athymic NUDE mice (Crl:NU(NCr)-Foxn1nu). After 3–4 weeks, the mice were euthanized, and teratomas were removed and fixed in 4% paraformaldehyde in PBS. Teratomas were cut into pieces with a 5 mm diameter, dehydrated, and paraffinized. The paraffin pieces were then cut into 5 μm slices using a microtome Leica RM2235 (Leica Biosystems, Wetzlar, Germany). The glass-slide attached paraffin slices were dried, rehydrated, and stained with hematoxylin, washed and then stained with eosin. Then they were washed, dehydrated, and mounted with Canadian balsam and coverslip. The teratoma histological sections were analyzed using the EVOS Cell Imaging Systems (Thermo Fisher Scientific, Waltham, MA, USA).

Organoids were fixed in 4% paraformaldehyde and gently spun down to a pellet (at 100 g). The supernatant was removed and organoids were resuspended in agarose gel and then embedded in paraffin. Paraffin-embedded organoids were then cut into 5-μm sections using a Leica RM2235 microtome (Leica Biosystems, Wetzlar, Germany), applied onto superfrost plus slides, and air dried at room temperature for 37 °C overnight. Samples were subsequently de-paraffinized then stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS)/Alcian blue, and immunocytochemistry was then performed. Antigen retrieval by 10 min of incubation in boiling 10 mM sodium citrate buffer (pH 6.0) was done for immunostained slides. Antibody information is presented in Additional file 2: Table S2. The morphometrics of sections stained with H&E or PAS was determined (Nikon Microphot FXA, Nikon Instruments Inc., Melville, NY) then analyzed using ImageJ (imagej.nih.gov; [20 (link)]) while immunofluorescence measurements of histological sections were recorded by confocal microscopy (Olympus Fluoview 1000, Olympus Corporation, Center Valley, PA).

The right lung inferior lobes were fixed with 10% formalin, embedded in paraffin, cut into 4-μm thick sections with a Leica RM2235 rotary microtome (Leica Biosystem, Germany) and mounted onto slides. After deparaffinization and hydration of the sections, they were stained with hematoxylin and eosin. Light microscopic examination was performed with a Nikon E400 microscope (Nikon Instrument Group, Japan).

Testicular organs of two Javan muntjacs were collected, subsequently fixed using an immersion fixation technique in Bouin's solution for 24 h, and then transferred to 70% ethanol as a stopping point. Prior to histological tissue preparation, testes were dissected into small pieces (0.5 × 0.5 × 0.5 cm). Testicular samples were then dehydrated in a graded ethanol series of 70%, 80%, 90%, and absolute, cleared in xylene, and immersed in paraffin infiltration, followed by embedding in a paraffin block. Paraffinized tissue was cut into 4-μm-thick sections using a Leica RM2235 manual microtome (Leica Biosystems, Nussloch GmbH, Germany) and placed on clean glass slides. Afterwards, paraffin was removed using a xylene solution, rehydrated, and subsequently stained with hematoxylin and eosin (H&E). All sections were dehydrated, cleared, and mounted with cover slips and Entellan® (Merck, Germany).

After 12 weeks of intervention, we administered BrdU at 20 mg/mL (100 mg/kg, Sigma) in sterile saline intraperitoneally for five consecutive days [23 (link),24 (link)]. Twenty-four hours after BrdU injection, all mice were anesthetized with 1% sodium pentobarbital (50 mg/kg, i.p.) and perfused with 50 mL of pre-cooled phosphate-buffered saline (PBS, pH 7.4) until the liver became white. The brains of mice were fixed with 4% paraformaldehyde, dehydrated with alcohol gradient, and made transparent with xylene. Then, according to the mouse brain atlas of Paxinos and Franklin “The Mouse Brain in Stereotaxic Coordinates,” the DG level representative area of the dorsal hippocampus was embedded in paraffin (1.22 mm/2.46 mm posterior–anterior to bregma). One hemibrain was serially cut into 4 μm thick sagittal sections using a paraffin microtome for immunostaining analysis (Leica RM2235, Leica Biosystems).