Ab91353

Immunostaining was applied on 5 μm thick paraffin sections. After deparaffinization and rehydration, the sections were treated by microwave for 20 min at 400 W in citrate buffer (pH 6.0). After antigen retrieval, samples were incubated for 1 hour at room temperature with primary antibody for nestin (Santa Cruz, USA, sc-23927, dilution 1:100). Sections were then treated with EnVisionTM Detection System (DAKO, Germany) using 3-amino-9-ethylcarbazole (AEC) as substrate, and counterstained with hematoxylin. Negative controls were performed by omitting the first antibody and stained by the EnVisionTM method, and for mouse monoclonal antibodies as isotype control mouse IgG1 (ab91353, Abcam, UK) antibody was also used. The slides were evaluated using a light microscope BX53 with DP12-CCD camera (Olympus, Germany).

Muscle samples were cut in a cryostat in 10 μm serial cross-sections, mounted on glass slides, and stained with previously characterized antibodies to demonstrate muscle fiber types and visualize the muscle cells' basement membrane. In brief, the sections were then incubated for 1 hour at room temperature with primary mouse IgM anti-MyHC I (A4.840, Developmental Studies Hybridoma Bank (DSHB) at the University of Iowa, Iowa City, IA, United States), includeed mouse IgG anti-MyHC IIA (SC-71, DSHB, and rabbit IgG anti-laminin (Z0097, Dako). Muscle sections were then washed in phosphate-buffered saline (PBS) and mounted using PermaFluor (Fisher Scientific). For visualization of capillaries, muscle cross-sections were fixed for 15 min in cold methanol at -20°C and rehydrated in PBS for 10 min at room temperature. A steamer achieved antigen retrieval using a citrate buffer (pH 6.0) for 20 min. Following quenching and protein blocking steps provided by the secondary antibody kit, slides were incubated with primary anti-CD31 antibody (ab9498) or IgG1 isotype control antibody (ab91353) from Abcam (Cambridge, United Kingdom) diluted 1:200 in PBS with 1% BSA overnight at 4°C. After three washes with PBS, secondary antibody incubation and HRP revelation were performed using the HRP/DAB Detection kit (ab64264; Abcam, Cambridge, United Kingdom) following the manufacturer’s instructions.

ChIP was performed using the EpiTect ChIP OneDay Kit (#334471, Qiagen, Hilden) according to the instructions of the manufacturer. In brief, 1.5x10⁶ BV-173 cells per sample were lysed and sonicated (5 times 6 s, 0.5 W) using the Vibra-Cell™ system (Sonics, Newtown, USA). Immunoprecipitation was performed with an anti-c-MYB antibody (ab17851, Abcam, Cambridge, UK) and an unspecific control IgG1 control antibody (ab91353, Abcam) according to the manual. For quantification of the immunoprecipitated DNA target, the ChIP qPCR Primer Assay GPH1003144(+)02A (Sabioscience/Qiagen) was applied employing the Roche LightCycler 480 System, using LC480 DNA Master SYBR Green and the standard LightCycler protocol as already described (Roche Diagnostics, Mannheim, Germany). Results derived from triplicate experiments were expressed as percentage of input sample taken before immunoprecipitation during the ChIP procedure. Therefore, the Ct values of the IP fractions were normalized to the input fraction.

Melanoma and nevi specimens were fixed in 10% buffered formalin and embedded in paraffin. Sections were deparaffinized and stained with H&E. For immunohistochemistry, deparaffinized sections were stained with the following primary antibodies for 1 hour at room temperature: anti-CCKAR polyclonal (PA3-116, 1:350; Thermo Fisher Scientific, Waltham, MA), anti-CCK polyclonal (2 μg/ml; ab27441, 1:100; Abcam, Cambridge, United Kingdom), rabbit IgG isotype control (2 μg/ml; ab37415, 1:250; Abcam), anti‒HMB-45 monoclonal (0.5 μg/ml; M0634, 1:50; Agilent Technologies, Santa Clara, CA), anti-Melan A monoclonal (2 μg/ml; M7196, 1:50; Agilent Technologies), and mouse IgG1 isotype control monoclonal (2 μg/ml; ab91353, 1:50; Abcam) antibodies. Immunoreactivity was detected using Histofine Simple Stain Max PO (Nichirei Biosciences, Tokyo, Japan). The color was developed using 3-amino-9-ethylcarbazole as the substrate.