Anti mitf antibody

Patient-derived melanoma sections of 14 primary tumors, 14 nodal tumors, and 6 distant metastases as well as pre-imaged, sequentially sectioned tumors stained using H&E were provided by the Ontario Tumor Bank of the Ontario Institute of Cancer Research (OICR, ethically collected after informed consent). All samples handled according to our ethical protocol HSREB#103381 (Western University and LHSC). Paraffin embedded sections were deparaffinized using xylene and sequential dilutions of ethanol. Antigen retrieval was performed in 1.5% of Vector Labs Antigen Unmasking Solution (Vector Laboratories, Burlington, ON, Canada) and heating in a microwave at 80% power. Sections were labeled for PANX1 (1:50; PANX1 CT-412) and a peptide pre-adsorption assay was performed as previously described [45 (link)]. A rabbit anti-MITF antibody (1:100; Abcam, Cambridge, UK) was used to identify melanoma cells. Alexa Fluor 488 goat anti-rabbit IgG (2 mg/mL; 1:400) was used as a secondary antibody and Hoechst 33342 (1:1000 in water) (Thermo Fisher Scientific) was used to label cell nuclei. Slides were mounted using VECTASHIELD^® mounting medium (Vector Laboratories) before imaging.

The Matrigel and Transwell chambers were obtained from BD Biosciences (San Jose, CA, USA) and Corning Costar (Acton, MA, USA), respectively. Antibodies against cleaved caspase-3, cleaved capase-9, PARP, Bax, Bcl-2, Bcl-xL, Fas, DR5, phospho-ERK1/2, ERK1/2, phospho-p38, p38, phospho-JNK, JNK, phospho-NF-κB, NF-κB, phospho-STAT3, STAT3, phospho-AKT, AKT, β-actin, rabbit IgG, and mouse IgG were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-MITF antibody was obtained from Abcam (Cambridge, MA, USA).