Anti mct1 mouse monoclonal antibody sc 365501

The data of 30 patients with SS were analyzed. Slides were heated for antigen retrieval in 10 mM sodium citrate (pH 6.0), followed by incubation with 1:200 anti-MCT1 mouse monoclonal antibody (sc-365501; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or isotype-matched control antibodies overnight at 4 °C. Immunodetection was performed using Histofine anti-mouse IgG (Nichirei, Tokyo, Japan) and the DAB substrate kit (Nichirei) according to the manufacturer’s instructions. Sections were counterstained with hematoxylin to create contrast. Sections were scored semi-quantitatively for cytoplasmic expression as follows: score 0, 0% immunoreactive cells; score 1, <5% immunoreactive cells; score 2, 5–50% immunoreactive cells; score 3, >50% immunoreactive cells. The intensity of staining was scored semi-qualitatively as follows: 0, negative; 1, weak; 2, intermediate; and 3, strong. The final score was defined as the sum of both parameters (extension and intensity) and grouped as negative (score 0 and 2) and positive (score 3–6), as previously described [26 (link)].

Total protein from cells (10 μg) or EVs (30 μg) was fractionated using an electrophoretic gradient across Mini-PROTEAN^® tris-glycine extended gels (BIO-RAD, Richmond, CA, USA). Loading samples were normalized according to the protein concentrations quantified using the Bradford assay. The gels were then transferred onto the Immun-Blot^® PVDF membrane (BIO-RAD) under wet electrophoretic conditions. The blotted protein was blocked for 1 h at room temperature with Odyssey^® blocking buffer in PBS (LI-COR, Lincoln, NE, USA) followed by incubation overnight at 4 °C with the following primary antibodies: 1:200 anti-MCT1 mouse monoclonal antibody (sc-365501; Santa Cruz Biotechnology); 1:200 anti-CD81 mouse monoclonal antibody (sc-23962; Santa Cruz Biotechnology); 1:2000 anti-tubulin hFAB Rhodamine (BIO-RAD). Thereafter, IRDye^® 800CW anti-mouse IgG secondary antibodies (LI-COR) were incubated with the protein-blotted membrane for 45 min at room temperature. Fluorescence was detected using the Odyssey^® imaging system (LI-COR).