Sc 6262

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.

Cells grown on coverslips were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 20 min at room temperature. For immunostaining, the cells were incubated with various primary antibodies at 37 °C for 1 h, washed three times with PBS and incubated with appropriate secondary antibody, anti-rabbit IgG conjugated with Dylight405 (711-475-152, 1:1,000, Jackson ImmunoResearch, West Grove, PA) or anti-mouse conjugated with Alexa488 (715-545-150, 1:1,000, Jackson ImmunoResearch) at 37 °C for 1 h. The coverslips were washed three times with PBS and incubated with anti HLA-DP monoclonal antibody conjugated with DyLight594 (H1584, 1:200, Leinco Technologies) at 37 °C for 1 h, washed three times with PBS and mounted on a slide with antifade medium (Vectashield, Vector Labs, Burlingame, CA). Fluorescence images were captured using a confocal microscope (Zeiss LSM700). The primary antibodies utilized were as follows: rabbit anti-Ii polyclonal antibody (sc-20082, 1:200, Santa Cruz Biotechnology), mouse anti-Ii monoclonal antibody (sc-6262, 1:200, Santa Cruz Biotechnology), mouse anti-EEA1 monoclonal antibody (610457, 1:250, BD Biosciences), mouse anti-LAMP1 monoclonal antibody (sc-20011, 1:100, Santa Cruz Biotechnology) and mouse anti-PDI monoclonal antibody (MA3-019, 1:500, Thermo Scientific).