Alexa 488 labeled streptavidin

After aldehyde quenching with 0.05M NH₄Cl and permeabilization with 0.1% Triton X-100, cells were preincubated with 1% bovine serum albumin (BSA) for 30 min. Successive incubation was performed for 48 h at 4°C in a mixture of anti-MAP2 and anti-GFAP primary antibodies and b-LEA in PBS/BSA 0.1%. This procedure was followed by incubation with a solution of PBS/BSA 0.1% containing the secondary antibodies DAM-Cy3 (polyclonal, anti-mouse IgG, made in donkey; 1:200 dilution; Cy3 fluorochrome; Jackson Immunoresearch Laboratories) and DAR-Cy5 (polyclonal, donkey anti-rabbit IgG conjugated to the indocarbocyanine Cy5, 1:200, Jackson Immunoresearch Laboratories) to reveal monoclonal and polyclonal antibodies, and Alexa-488-labeled streptavidin (1:200, Molecular Probes) for b-LEA lectin cytochemistry. After rinsing, samples were mounted on coverslips with PBS/glycerol and inspected with a TCS-NT (Leica Laserteknik GmbH) laser scanning confocal microscope, to visualize triple fluorescent labeling. The original emission color of fluorochrome conjugated to secondary antibody DAR-Cy5 has been set to blue to facilitate visual inspection of confocal images. All the thickness of cell cultures (about 12 μm) was acquired by digital superimposing of at least 10 serial optical sections.

For the analysis of MGL ligand expression the previously described MGL-Fc [12 (link)] was used. FITC-labeled anti-human IgG-Fc antibody (Jackson Immunoresearch, Suffolk, UK) was used to detect binding of MGL-Fc by flow cytometry. Biotinylated lectin Helix pomatia agglutinin (HPA) was obtained from Sigma–Aldrich (st. Louis, MO). Extracellular MGL ligand expression was analyzed by flow cytometry as reported before [44 (link)]. Binding of HPA (5 μg/ml) was analyzed by incubating cells for 30 min at 37°C with the lectin, followed by staining with Alexa-488 labeled streptavidin (Molecular Probes, Waltham, MA) and analyzed on a FACSCalibur (BD Biosciences, Franklin Lakes, New Jersey). Cells were selected based on forward-scatter and side-scatter patterns and exclusion of dead cells by 7-amino-actinomycin D (Molecular Probes) staining. Intracellular phosphorylated-ERK1/2 levels were analyzed by fixing with 2% paraformaldehyde and permeabilizing with 90% methanol at 4°C, followed by incubation for 1 hour at room temperature with FITC-coupled rabbit-anti-phospho-ERK1/2 (Thr202/Tyr204) (Cell Signaling), followed by analysis on a FACSCalibur.