Immunofluorescence detection of microglial markers
The MAC-1/CD11b antibody (clone M1/​​70.15) from BioRad (Oxford, UK) and the rabbit anti-ionized antibody of the calcium-1 binding molecule (Iba-1, #019-19741) from Wako Chemicals (Neuss, Germany) were used to monitor microglial cell responses at the cellular level. Briefly, cultures were fixed with 4% formaldehyde in PBS (20 min, at room temperature), washed and incubated sequentially with antibodies against MAC-1/CD11b (1:1000 in PBS for 72 hours) and Iba-1 (1: 500 in PBS-0.2% Triton X-100 overnight). Immunodetection of MAC-1/CD11b and Iba-1 were performed with anti-rat Alexa-Fluor 555 and anti-rabbit Alexa-Fluor 488 secondary antibodies, respectively (Invitrogen). Nuclear counterstaining with Hoechst 33342 (1 μg/ml for 5 min) was performed to enable automated focus during the cell counting procedure with an Arrayscan XTi workstation (Thermo Fisher Scientific, Courtaboeuf, France). Fluorescent images were automatically acquired with a 20x objective, and variations of immunofluorescence signal intensities were quantified at the cellular level using the HCStudio software. For each treatment condition, at least 2,500 cells were processed for image analysis.