Sc 20062

For the analysis of CD74 cell surface expression, cells were incubated with monoclonal antibody to CD74 (sc-20062 or sc-6262; both from Santa Cruz), monoclonal antibody to MET (MAB3582; R&D Systems, Wiesbaden, Germany) or the respective isotype control (MAB002; R&D Systems), followed by incubation with a phytoerythrin (PE)-conjugated F(ab′)₂ fragment (115-116-071; Dianova). For the analysis of primary lymphoid cells, indirect staining for CD74 was performed in a first step as described above, followed by incubation with APC-labeled anti-CD19 (C7224; Dako, Hamburg, Germany) or anti-CD4 (IM2468; Beckman Coulter, Krefeld, Germany) antibodies. Immunofluorescence was analyzed using a FACSAria flow cytometer and CELLQuest software (Becton Dickinson). The percentage of viable and apoptotic cells was determined by Annexin V-FITC/propidium iodide (PI) double staining (Bender MedSystems) and flow cytometry using a FACSAria flow cytometer. Cells double negative for Annexin V-FITC and PI were considered as viable cells. For light microscopy, a Leica CTR6000 microscope equipped with a Leica DFC350FX camera was used.

The detection of CD74 protein in formalin-fixed and paraffin-embedded tissue sections was performed employing the anti-CD74 antibodies sc-20062 and sc-6262 (both Santa Cruz) at a dilution of 1:1000 after a 20 min treatment in EDTA buffer (Retrieval solution 2; EDTA-buffer pH 8.8 at 98 °C for 20 min). Bound antibody was visualized using the alkaline phosphatase anti-alkaline phosphatase method and FastRed as chromogen (DAKO) or, after peroxidase blocking, DAB-chromogen. For c-MET detection, the prediluted anti-total c-Met (clone SP44) rabbit monoclonal antibody was obtained from Ventana (Ventana Medical System). Immunostaining was carried out according to the manufacturer´s protocol on the BenchMark Ultra platform from Ventana utilizing the ultraView detection kit.