E600 microscope

Transfected MDCK cells were trypsinysed, washed twice in PBS, diluted in DMEM and suspended as hanging drops from the lid of a 24 well culture dish. Wells were filled with sterile water to prevent drops drying (Thoreson et al., 2000 (link)). Culture dishes were kept in a humid 5% CO₂ incubator at 37°C and cell aggregation was determined 4 h after plating. Cells in each drop were passed 10 times through a standard 200 μl Gilson pipet tip and photographed through a Nikon E600 microscope, using a 20X phase contrast objective. The number of isolated cells and cells forming aggregates were counted in five random fields from three independent experiments.
Adhesion assays were lightly modified from previously described (Orozco et al., 1983 (link)). Briefly, MDCK cells in suspension were mixed with washed erythrocytes (1:100 ratio). Cell mixture was incubated for 0.5, 1 and 2 h at 37°C, fixed with 2.5% glutaraldehyde for 30 min at 37°C and washed three-times with PBS. Erythrocytes were counterstained with 4.5 mM diaminobenzidine for 30 min at 37°C. Finally, adhered erythrocytes to each MDCK cell in ten random fields from three independent experiments were counted through a Nikon E600 microscope.
For inhibiting cell aggregation and erythrocytes adhesion, before experiments, 2 × 10⁵ MDCK cells were incubated with 10 μg mAbAdh antibody or IgM isotype for 30 min at 37°C.

Area fractions of GFAP+ astrocyte elements in multiple regions of the hippocampus were measured, as described in our previous study [50 (link)]. This quantification was performed using Image J for Windows. Briefly, gray-scale images of different regions of the hippocampus were digitized using a 20X lens in a Nikon E600 microscope outfitted with a digital video camera attached to a computer. A binary image was created from each image by choosing a threshold value that retained all GFAP+ elements but no background. The area occupied by the GFAP+ structures in the binary image was then quantified through “Analyze” function in Image J. The area fraction of GFAP+ structures were calculated for the entire hippocampus by using data from all selected serial sections.