Sc 2777

The procedure for immunofluorescence (IF) was performed as previously described [16] (link). Briefly, EL4 cells transfected overnight with pEGFP-C3-siOCILRP2 were stimulated with anti-CD3/CD28 antibodies. For the other groups, EL4 cells were stimulated with or without anti-CD3/CD28 or anti-CD3/CD28/OCILRP2 antibodies. All cells were incubated on glass cover slips and fixed with 3.5% paraformaldehyde for 15 min, which was then stopped by the addition of 30 mM glycine. After washing, the cells were permeabilized with 0.1% Triton X-100 for 15 min and blocked with 3% bovine serum albumin in PBS for at least 1 h (4°C). A polyclonal anti-OCILRP2 antibody (AF3370, R&D systems, USA) and monoclonal anti-DAP12 antibody (sc-133174, Santa Cruz, USA) were used at a 1∶2000 dilution. Goat anti-mouse-IgG-PE (sc-3738, Santa Cruz, USA) and rabbit anti-goat-IgG-FITC (sc-2777, Santa Cruz, USA) secondary antibodies were used at a 1∶100 dilution. Nuclei were stained with 4′, 6′-diamidino-2-phenylindole dihydrochloride (DAPI) (D9542, Sigma-Aldrich, USA), and the cells were examined by confocal microscopy (98DDFR/470111CR, Bio-Rad, USA).

The brain was excised after the mice were decapitated. Each SNC was fixed in 4% paraformaldehyde and each hypothalamus was processed for paraffin embedding and sectioned into 5.0-μm sections. Samples were incubated with primary antibodies overnight and with secondary antibodies conjugated to FITC or rhodamine for 2 hours (sc2777and sc2092, respectively; Santa Cruz Biotechnology, Santa Cruz, CA). The DAPI stain was used for nuclear staining while the Leica FW 4500 B microscope captured the images. Hypothalamic areas were observed according to the landmarks in the mouse brain atlas [29 (link)]. Analysis and documentation of the results were performed using Leica Application Suite V3.6 (Switzerland).

IL6 protein expression was investigated by immunostaining and fluorescence microscopy 48 hours after induced macrophages were put in contact with M1—M5 materials. Briefly, cells in matrices were fixed with 4% paraformaldehyde for 2 hours and then permeabilized for 1 hour with a 0.1% Triton X100 solution in 2% BSA. The systems were further immunostained overnight using a primary anti-IL6 antibody (sc-1266, 1 : 100 dilution, Santa Cruz, USA) and then secondary antibody conjugated with FITC (sc-2777, 1 : 100 dilution, Santa Cruz, USA) for 1 hour at 4°C. Cell nuclei were stained with DAPI. Staining was visualized in an Olympus IX73 fluorescence microscope.