Fig. 4. Double labelling of inhibitory motor neurons through combination of backfilling nerve 6 (A; red fluorescence, Cy3) and anti-GABA immunocytochemistry (B; green fluorescence, Cy2). (Ai,Bi,Ci) A whole-mount preparation (anterior is to the top; ganglion midline indicated by dotted line, backfill labels are contralateral to the side of nerve 6 exit); (Aii,Bii,Cii) a cross section through the same area of the prothoracic ganglion in a different animal (ventral is to the top). The overlays in Ci and Cii show which neurons have acquired both labels (arrowheads indicate the same cells in Ai–Ci, and Aii–Cii, respectively). Note that the cell marked with an asterisk in Ci does not stain for GABA in Bi. It only appears orange because it is located on top of cells labelled in green. Scale bar, 50 µm.