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Fig. 4. VGAT (red A,B,D,F) co-localizes with glucagon (green C) in A-cells. Insulin-positive (green A) and GABA-positive (green D) core cells, are very slightly labelled for VGAT (red D). Synaptophysin (green E) is in both A- and B-cells. Electron microscopic immunogold labelling with three different VGAT antibodies (G,H,I) shows strong signal in the SGs of A-cells (G,H,I) but not B-cells (G). The identity of the A-cell SGs is confirmed by double labelling (H) with 10 nm gold particles for glucagon and 15 nm particles (red arrows) for VGAT. Insets: higher power photographs of SLMVs (arrowheads) labelled for VGAT, from the two cell types. Electronmicroscopic quantification (J) shows that VGAT immunogold density over A-cell SGs is >10-fold higher than that over B-cell SGs, or those over mitochondria or cytoplasm of either A- or B-cells (P<0.01). Further analysis (K,L) of the distribution of VGAT was conducted as shown in Fig. 1 (see also Materials and Methods). The density of VGAT immunogold associated with the membrane of SGs (K) is only slightly higher in A-cells than in the B-cells, but is significantly higher than cytosol background (P=0.01 in both A- and B-cells, n=5). Analysis of the distribution of VGAT in the residual cytoplasm (free of SGs and mitochondria) shows a many times higher density of VGAT immunogold associated with SLMVs than over cytosol (L) (P<0.001 in A-cells, P=0.007 in B-cells, n=5), which is close to the background noise over tissue-free plastic (2-3 part/µm², not subtracted). SLMV-associated VGAT relative to cytosol background is slightly higher in A-cells than in B-cells (P=0.008, n=5). Scale bars G-I: 200 nm (scale for insets: 15 nm gold particles).

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