Hoechst no 33258

In order to detect LC3 II immunoreactivity in neurons and astrocytes at 10, 20 and 30 days of culture, double immunofluorescence was performed using LC3 antibody together with cell type markers. In details, primary cultures were fixed with 4% paraformaldehyde at room temperature for 20 min. After fixation, cells were washed with PBS, then permeabilized with 0.1 % Triton X-100 for 10 min, treated with 5% BSA buffer for 30 min to block endogenous nonspecific sites and incubated overnight with one of the following monoclonal primary antibodies: anti-GFAP diluted 1:500, and anti-III β-tubulin, diluted 1:200. In addition, a polyclonal anti-LC3 II antibody, diluted 1:100 was used.^{33 (link)} Cells were then incubated with a mixture of two secondary antibodies anti-rabbit TRITC, diluted 1:100, and anti-mouse FITC, diluted 1:100. All antibodies were from Sigma. All samples underwent blue nuclear counterstaining for fluorescence microscopy with Hoechst no. 33258 (Sigma-Aldrich). Finally, labeled cultures were mounted with FlourSaveTM Reagent (Calbiochem, San Diego, CA, USA). Cultures were observed and photographed using a Leica TCS confocal microscope with excitation and emission filters 488 and 527 for FITC, 543 and 587 for TRITC, and 365 and 448 for Hoechst blue no. 33258.

The mouse RAW 264.7 macrophage cell line was purchased from the American Type Culture Collection (ATCC). The cells were cultured overnight on poly-L-lysine-coated cover glass (Sigma-Aldrich Co.) in 12-well plates (Nunc Inc.). The FITC-labeled S. aureus MW2 was treated with PBS or IgM for 1 h, and then, the bacteria were added to the plate cultured with RAW 264.7 cells. The cells were incubated for 1 h and fixed with 4% paraformaldehyde (Affymetrix Inc.). The fixed cells were washed with PBS, and then, Hoechst No. 33258 (Sigma-Aldrich Co.) was added at room temperature. The mounted cells were analyzed using a LSM 710 laser scanning microscope (Carl Zeiss Co.) previously described (18 (link)). The phagocytic index was measured by counting the number of FITC-labeled S. aureus MW2 phagocytosed by RAW 264.7 cells as previously described (18 (link)).
Additional Material and Methods are provided in the Supplementary Material.