Air confocal laser microscope

Macrophages were cultured on sterilized glass coverslips in 24-well plates and infected with Mm for 4 h or treated as if infected (MOCK), as the method mentioned in section 2.4. Cells were fixed with 2% paraformaldehyde for 30 min at 4°C, permeabilized with 0.1% Triton X-100 for 15 min, blocked with 5% BSA in TBST for 30 min, and stained with rabbit anti-FBXW7 antibody and mouse anti-NF-κBp65. Following the primary antibody, the samples were stained with FITC-labeled anti-rabbit secondary antibody and Cy3-labeled anti-mouse secondary antibody. DAPI (4, 6-diamidino-2-phenylindole; Sigma) was used to stain the nuclei. The co-localization of FBXW7 and NF-κBp65 was detected by imaging with a Nikon AIR⁺ confocal laser microscope. The signal for nuclear p65 was determined by counting the number of nuclei (DAPI) merged with p65, dividing by the total number of nuclei scored. At least 200 total nuclei per well were counted in each of the triplicate wells.

Both primary and BV-2 microglial cells, control and treated, were washed with 1× phosphate-buffered saline (PBS) thrice and were fixed with acetone to methanol followed by permeabilization with 0.3 % Triton X-100 in PBS (PBST). Cells were then incubated with anti-α-tubulin (1:500) or anti-NF-kB (1:300) or anti-AP1 (1:300) or anti-HSP-70 (1:300) (from Sigma-Aldrich) diluted in 2 % BSA, for 24 h at 4 °C in humid chamber. After two to three washings with 0.1 % PBST, the cells were incubated with the secondary antibody anti-mouse IgG 488 and anti-rabbit IgG 488 (prepared in 2 % BSA (1:500)) for 2 h at room temperature. Cells were incubated with (DAPI, 1:5000 in 1× PBS) for 10 min for nuclear staining and then mounted with anti-fading reagent (Fluoromount, Sigma). For CellRox and Mitotracker staining, after treatment period, the dye was added in culture according to the manufacturer’s instructions; cells were then fixed and mounted. Images were captured using Nikon AIR Confocal Laser Microscope and analyzed using NIS elements AR analysis software version 4.11.00. Experiment was performed in triplicate.

The control and treated cells were washed with 1X PBS thrice, fixed with 1:1 acetone and methanol followed by permeabilization with 0.3% Triton X-100 in PBS (PBST). After blocking with 2% BSA in PBS, cells were incubated with anti-MAP2 (1:200, Sigma-Aldrich) monoclonal antibody diluted in blocking solution for 24 h at 4 °C in a moist chamber. Then the cells were washed twice or thrice with 0.1% PBST followed by incubation with anti-mouse IgG 488 secondary antibody (diluted at a concentration of 1:500 in 2% BSA) for 2 h at room temperature. For nuclear staining, cells were incubated with 1:5000 dilution of DAPI in 1X PBS for 10 mins. Cells were then mounted with anti-fading reagent (Fluoromount, Sigma-Aldrich). Nikon AIR Confocal Laser Microscope was used for capturing the images. Experiments were performed in triplicate. For quantifying MAP2 relative optical intensity, ROIs were selected from different cells (at least 100 cells per group) and analyzed for the intensity using NIS elements AR analysis software version 4.11.00. For neurite outgrowth analysis, at least 100 cells per group were measured for length of neurites using the Image Pro Plus software (Media Cybernetics, Silver Spring, USA) and percentage neurite outgrowth per group was calculated using Microsoft excel and Sigma Stat for windows (version 3.5).