S9226

Four wild male Sunda porcupines were obtained from Central Java, Indonesia. The animals were euthanized by an overdose injection of ketamin HCl (10–15 mg/kg) and xylazine HCl (0.10–0.15
mg/kg) administrated intramuscularly before taking organ samples. The removed testes were immediately fixed in Bouin’s fluid for 24 hr. After fixation, the tissue samples were dehydrated in
a graded series of ethanol, cleared in xylene and embedded in paraffin. The samples were cut serially at 4 µm thickness and mounted on aminopropyl-triethoxy-silane-coated
slides (S9226, Matsunami Glass, Osaka, Japan). After deparaffinization, the testicular tissue sections were stained with hematoxylin and eosin (HE) to examine the general structure. All
experimental procedures were authorized by the Ethics Committee for the Use of Animals at Gadjah Mada University (Number: 0014/EC-FKH/Eks/2017). Collection permit (number:
SK.56/KSDAE/SET/KSA.2/2/2018) was issued by the Directorate General of Natural Resources and Ecosystem Conservation (Direktorat Jenderal Konservasi Sumber Daya Alam dan Ekosistem, Jakarta,
Indonesia) under the Ministry of Environment and Forestry of the Republic of Indonesia.

Following completion of each experiment, mice were deeply anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg), perfused intracardially with 4% paraformaldehyde phosphate‐buffer solution and decapitated. Brains were removed from the skull and postfixed in the same fixative overnight. Subsequently, brains were cryoprotected in 20% sucrose overnight, frozen, and cut at 25 μm thickness on a cryostat (Leica CM3050 S; Leica Biosystems, Wetzlar, Germany). Sections were mounted on silane‐coated glass slides (S9226; Matsunami Glass, Osaka, Japan). Sections were incubated with the primary antibodies overnight at room temperature. The following antibodies were used: anti‐green fluorescent protein (GFP) (1:200, goat polyclonal; Rockland Immunochemicals, Pottstown, PA, USA). For fluorescence microscopy, sections were treated with species‐specific secondary antibodies conjugated to Alexa Fluor 488 (1:1000; Invitrogen, Carlsbad, CA, USA) and DAPI (1 mg/mL; Sigma‐Aldrich, Saint Louis, MO, USA) for 2 h at room temperature. Fluorescence images were obtained using an all‐in‐one microscope (BZ‐X710; Keyence, Osaka, Japan).