Fv10i scanning confocal microscope

Mice were deeply anesthetized with pentobarbital and transcardially perfused with PBS and then 3.2% paraformaldehyde. Brains were postfixed in 3.2% paraformaldehyde for 48 h. Fixed brains were sectioned (40 μm) using a vibratome (Leica). Sections were blocked with 3% goat serum. Sections were washed and incubated with a rabbit anti-mouse ionized calcium binding adaptor molecule 1 (Iba-1) antibody (Wako Chemicals, USA), overnight at 4°C, followed by incubation with the appropriate fluorescent-conjugated secondary antibody (Invitrogen). Slices were then mounted on glass slices with Fluoroprep (Dako) containing 1 mg/ml Hoescht, a fluorescent specific DNA dye (Invitrogen). Each fluorochrome was independently captured with an FV10i scanning confocal microscope (Olympus, France). Quantification of Iba-1 staining was performed using ImageJ software (NIH). The software generated fluorescence intensity values by tracing the region-of-interest (ROI). Arbitrary units were defined in terms of strength of fluorescent signal. The final intensity values were calculated by subtracting the area of the selected region multiplied by the background fluorescence from the fluorescence intensity of the region-of-interest (ROI): fluorescence intensity (arbitrary units) = fluorescence intensity of ROI − (area of selected region × mean fluorescence of background).

BV2 cells were grown on poly-D-lysine-coated glass coverslips. After treatment, cells were fixed with 3.2% paraformaldehyde, then permeabilized in PBS pH 7.4, containing 0.3% Tween and 3% BSA. Cells were then incubated overnight with the appropriate primary antibody directed against CD11b (Abcam), iNOS (Merck Millipore), AdipoR1 or AdipoR2 (Sigma Aldrich) in the same buffer. At the end of the incubation time, cells were washed three times with PBS and incubated with either Alexa488-conjugated or Alexa594-conjugated secondary antibodies (Invitrogen) and Hoechst fluorescent stain (Invitrogen) for 1 h at room temperature. For NF-κB p65 subunit nuclear translocation study, cells were pre-treated with 10 μg/ml gApN or saline for 1 h before addition of 0.5 μg/ml LPS for 3 additional hours. Cells were then fixed and permeabilized as previously. Primary antibody against p65 was from Cell Signaling. Images were captured with an FV10i scanning confocal microscope (Olympus, France) and analyzed using Image J software (“National Institutes of Health” Image J Software™).