Goat anti gpnmb antibody

A 1 mm tissue cores were collected from 30 normal, 72 pre-neoplastic lesions and 43 cancers and used to build Tissue Microarrays (TMAs) in this study. Positive and negative non-colon controls were also spotted on the TMAs. Slides were cut from the generated TMAs. De-paraffinization/hydration of the TMAs was performed as follows: two xylene (5 min each), followed by two 100% ethanol washes (5 min each), followed by 95% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, followed by H₂O and a TBST wash for 5 min on a shaker. Immunohistochemical staining was done according to standard procedures using a polyclonal goat anti-GPNMB antibody (1:500 dilution; R&D Systems) and a HRP-conjugated donkey anti-goat secondary antibody (1:500 dilution; Jackson ImmunoResearch Laboratories; West Grove, PA). Sections were developed with 3–3-diaminobenzidine-tetrahydrochloride (DAB) and counterstained with hematoxylin. The immunostained slides were evaluated by two expert gastrointestinal pathologists (E.L, B.S). Both intensity and percentage of staining were used to evaluate and compare samples.

Immunoprecipitation assays were performed using a Classic IP Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. NSC34 cells were plated at 1.4 × 10⁵cells/well in 6-well plates (BD Biosciences) and incubated overnight. The cell lysates were incubated with mouse anti-NKA α1 antibody, mouse anti-NKA α3 antibody, or normal mouse IgG (Santa Cruz Biotechnology). A mixture of equal parts of a protein sample and sample buffer with 20% 2-mercaptoethanol (Wako, Osaka, Japan) was subjected to SDS-PAGE (Wako). The separated proteins were then transferred onto a polyvinylidene difluoride membrane (PVDF, Immobilon-P; Merck KGaA, Darmstadt, Germany). To visualize the proteins on the membrane, the primary antibody was goat anti-GPNMB antibody (1: 500, R&D Systems Inc., Minneapolis, MN, USA) and the secondary antibody was horseradish peroxidase (HRP)-conjugated rabbit anti-goat antibody (1: 2000, Thermo Fisher Scientific). The immunoreactive bands were visualized using a chemiluminescent substrate (ImmunoStar^® LD; Wako). The band intensity was measured using an imaging analyzer (LAS-4000; Fuji Film, Tokyo, Japan).