Fig. 4.

ABHD6 immunoreactivity in hippocampal neurons. HEK293 cells were transiently transfected with HA-tagged mABHD6 and stained using HA (1:500) and ABHD6 (1:2000) antibodies. A, DAPI staining identifies cell nuclei (arrowheads). B, HA antibody (detected with Texas Red secondary) staining shows HA-mABHD6 transfected cells (nuclei indicated by arrows). C, mABHD6 staining (FITC) labels the same cells identified with the HA antibody. D, Overlay of HA- and mABHD6-images shows both antibodies overlap completely, DAPI fluorescence identifies all cells in the field. The nontransfected cells (which endogenously express ABHD6) are weakly labeled with the mABHD6 antibody. Scale bar, 15 μm. E1 and E2, left, ABHD6 staining of an autaptic culture; right, ABHD6 antibody preincubated with immunizing protein (5 μg/ml) Scale bars, 25 and 50 μm. F, left, circular ABHD6 staining commonly found on autaptic islands. Note that the island (cells visible in differential interference contrast microscopy) extends beyond the edge of the staining. Scale bar, 50 μm. G, ABHD6 (red) and glutamine synthetase (Glut Synth, green) staining are mutually exclusive (arrows). Scale bar, 7.5 μm. H, an example of ABHD6 positive neuronal island with no glia positive for glutamine synthetase. Scale bar, 10 μm. I) ABHD6 (red) does not colocalize with SV2 (green). Scale bar, 7.5 μm. J, ABHD6 (red) does colocalize with some dendritic spines labeled by MAP2 (green, overlap in yellow). Top, overlap; middle and bottom, MAP2 and ABHD6, respectively, with arrows to identify several points of overlap. Scale bar, 7.5 μm.