Tyrosine hydroxylase

The protocol for immunohistochemistry as previously described.⁴⁷ The retinas were incubated in PBS with 1% Triton X-100 and 0.5% Tween 20 for 1 h at room temperature and in 4% BSA for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies: RBPMS (1:500, Abcam), ChAT (1:100, Abcam), VGlut2 (1:100, Frontier Institute), syntaxin (1:100, Abcam), tyrosine hydroxylase (1:100, Abcam) and Prox1 (1:100, AngioBio) in blocking buffer. Secondary anti-rabbit, mouse, and goat IgG, conjugated with Alexa TM488, TM594, and 633, respectively (1:1,000; Molecular Probes), were applied for 1 h at room temperature. The retinas then were flat mounted, and the sections were mounted on slide glass. The TUNEL assay was performed based on our previous reports.⁴⁸ (link)^,49

eWAT was harvested using the Adipose Protein Extraction Kit (MinuteTM) for protein extraction. Eighty milligrams of adipose tissue was homogenized with 250 ml of extraction buffer. After centrifugation at 2,000 g for 1 min, the tissue was transferred to a filter cartridge with a collection tube and incubated at -20°C for 20 min. After incubation, the tissue was immediately centrifuged at 2000 rpm for 1 min with the cap open. The flow-through contained total proteins from the adipose tissue. Then, 30 mg of total tissue lysate was separated by SDS-PAGE (10% separation gel and 5% concentration gel). Electrophoresis was performed at 70 V for 0.5 h and 110 V for 1 h. The protein bands were transferred to a PVDF membrane using e-Blot (Genscript), and 5% BSA was added for 1 h to block the membrane. The membrane was then made to react with primary antibodies against ATGL (1:1000, Abcam), HSL (1:3000, Abcam), p-HSL (1:1000, Sab), NLRP3 (1:1000, Abcam), Caspase-1 (1:1000, Abcam), IL-1β (1:1000, Abcam), MAOA (1:1000, Abcam), β-actin (1:1000, Abcam), and tyrosine hydroxylase (1:1000, Abcam) at 4°C overnight. Incubation with the corresponding secondary antibodies (diluted to 1:3000, Abcam) was performed at room temperature (18°C -25°C) for 1 h.

The constructed paraffin-embedded tissue blocks were cut in 5-μm-thick sections to perform H&E staining, Masson’s trichrome staining and immunohistochemical staining. Immunohistochemical staining was performed using the Ventana BenchMark XT automated stainer (Ventana, Tucson, AZ). The primary antibodies of neurofilament (1:50, MS-359-S1; Thermo Fisher Scientific, Cheshire, UK), tyrosine hydroxylase (1:100, Abcam, Cambridge, UK), nNOS (1:100, sc648; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Ki-67 (1:100, Abcam, Cambridge, UK) were performed. A Ventana UltraView DAB detection kit was applied, and the slides were counterstained with hematoxylin, dehydrated, and mounted. The determined criterion for the main and minor branches of the DPNs were the sizes of the nerve branches. In fact, the sizes of the main branches of the DPNs were similar to those of the dorsal artery. Therefore, if the nerve branches were smaller than the dorsal artery at the dorsal side of the penis between the tunica albuginea and the Buck’s fascia, it was determined to a minor branch of the DPNs.