The Journal of Neuroscience, July 26, 2006, ():
Expression and Function of SNAP-25 as a Universal SNARE Component in GABAergic Neurons
J. Neurosci. Tafoya et al.
26: 7826
Supplemental data
Files in this Data Supplement:
- supplemental material
-
Supplemental Figure 1: SNAP-25 immunoreactivity found in both GABAergic and glutamatergic puncta within the hippocampus. A, B, Using data acquired from dual stain immunofluorescence of adult hippocampal slices from Figure 6, SNAP-25, VGAT, and VGLUT1 staining patterns were reanalyzed to visually assign a pixel intensity threshold that isolated focal accumulations defined as puncta (left panel, red signal) from other immunofluorescently labeled structures of the tissue (left panel, gray signal). The area of a representative single pixel clusters (12 pixels x 12 pixels or 144 pixels squared; left panel, white circle) was selected to define the lower size limit of immunofluorescent puncta. To quantify the colocalization of the transporters with SNAP-25, these criteria were assigned for both VGAT and VGLUT1 immunostaining (left panel, A and B, respectively). Those puncta that overlapped with SNAP-25 (green, middle panels) were subtracted using NIH ImageJ analysis software. The very few clusters representing either VGAT or VGLUT1 stain that did not colocalize with SNAP-25 (right panel, white arrows). C, Statistical analysis revealed that SNAP-25 is found in ~95% of either VGAT- (black bar) or VGLUT1-positive puncta (gray bar). In contrast, analysis of the puncta resulting from VGAT and VGLUT1 dual staining showed little, if any, colocalization (white bar). Sections were imaged by laser confocal microscopy as described in the Materials and Methods using a 63X objective (optical slice 0.81 μm) and with digital magnification to 420X. The histogram represents average values (error bars, SEM) obtained from nine images taken from three animals (***, p<0.001).
