Ab210546

Quadriceps femoris (QF), tibial anterior (TA) and extensor digitorum longus (EDL) were fixed in 10% formalin. After hematoxylin and eosin staining, using ImageJ, diameters of muscle fibers were measured in 30 randomly selected fibers per mouse^{13 (link)}.
Skin of UV-irradiated areas was retrieved at 36 weeks of age, and fixed in 10% formalin. After hematoxylin and eosin staining, the thickness of epidermis and dermis in ten different fields was measured under a light microscope (magnification × 100)^{34 (link)}. For immunohistochemical analysis of skin damage, additional tissue sections were stained with rabbit anti-Melan-A monoclonal antibody (ab210546, 1:100 dilution; Abcam, Cambridge, MA, USA) to evaluate melanocytes or keratinocytes with melanin pigmentation, rabbit anti-Cleaved Caspase-3 antibody (ab2302, 1:500 dilution; Abcam, Cambridge, MA, USA) to evaluate apoptosis. These antibodies were confirmed to cross-react with mice antigens with a datasheet provided by the manufacturer. Cells undergoing apoptosis were also identified by the TdT-mediated dUTP nick-end labeling (TUNEL) method with a kit (Apoptosis in situ Detection KIT Wako, FUJIFILM Wako Pure Chemical Co., Osaka, Japan). The staining procedure was carried out according to the manufacturer’s recommendation.

Thirty‐six representative specimens including all JMN (n = 12), part of ALMs (n = 12), and ILNMs (n = 12) among IHC‐positive staining tissues were sampled to perform the multi‐immunofluorescence assays. Formalin‐fixed paraffin‐embedded slices (4‐µm thickness) were deparaffinized and antigen unmasked. Then the specimens were blocked with 10% donkey serum at room temperature (RT) for 30 min and were incubated with first primary antibody of anti‐OPN3 (1:100; MD4034‐100; MDL) overnight at 4°C and secondary antibody marked with HRP incubate at RT for 50 min in the dark condition. Next, the sections were stained with cyanine 3 (CY3)‐tyramide signal amplification (TSA) solution (all from Siwega) at a 1:500 dilution for 10 min at RT in the dark. The slides were again immersed antigen retrieval buffer (EDTA) via microwave antigen repair method. Repeat the above steps, the slides were stained with the second primary antibody of anti‐Melan A (rabbit 1:1000; ab210546; Abcam), corresponding secondary antibody and CY5‐TSA (all from Siwega), respectively. DAPI (C0060; Solarbio) incubation at a 1:1000 dilution at RT for 5 min was used for nuclei staining.

Immunofluorescence (IF) was performed on sectioned paraffin-embedded tumor constructs or fixed cells cultured for 3–7 days on X‐tra^® slides to analyze biomarker presence of SF-1 (ab240394, Abcam), Melan A (ab210546, Abcam), inhibin-α (ab47720, Abcam), IGF-2 (ab226989, Abcam), and β-catenin (ab ab223075, Abcam). Nuclei were stained with DAPI (P36962, ThermoFisher). The slides were imaged with Nikon A1R confocal microscope at 40 × magnification at room temperature.