Sc 59079

For the immunofluorescence analysis, HEK 293 cells were transfected with either control siRNA or siRNAs targeting HNRNPC as described under Cell Culture and RNAi, 48 h post transfection cells were fixed with 4% paraformaldehyde for 30 min, permeabilized, and blocked with PBS containing 1% BSA and 0.1% Triton X-100 for 30 min. Primary anti-CD47 antibody (sc-59079 from Santa Cruz Biotechnology) was incubated for 2 h at room temperature at a dilution of 1:100 in the same buffer. To visualize CD47 in cells, secondary antibody conjugated with Alexa Fluor 488 was applied, while the nucleus was labeled with Hoechst dye. Imaging was performed with a Nikon Ti-E inverted microscope adapted with a LWD condenser (WD 30mm; NA 0.52), Lumencor SpectraX light engine for fluorescence excitation LED transmitted light source. Cells were visualized with a CFI Plan Apochromat DM 60× lambda oil (NA 1.4) objective, and images were captured with a Hamatsu Orca-Flash 4.0 CMOS camera. Image analysis and edge detection was performed with NIKON NIS Elements software version 4.0. All images were subsequently adjusted uniformly and cropped using Adobe Photoshop CS5.

FACS analyses of siRNA transfected cells were performed similar to immunofluorescence studies (see above) except that cells were not permeabilized prior to the treatment with antibody against CD47 (sc-59079 from Santa Cruz Biotechnology). Analysis of Alexa Fluor 488 signal and counts was carried out on a BD FACS Canto II instrument, and data were analyzed with the FLOWJO software. An equal pool of siRNA samples from each transfection set was mixed for the IgG control staining to rule out nonspecific signals.