Figure 4: Ultrastructural localization of Hh in imaginal discs.

Correlative light-electron microscopy of wing discs expressing Hh-GFP. Discs were labelled ex vivo with a rabbit anti-GFP antibody and then fixed and processed for cryosectioning. Ultra-thin sections, cut orthogonally to the V/D axis, were incubated with a fluorescent (Alexa594) 1.4-nm nanogold-conjugated anti-rabbit Fab’ probe. Labelled cryosections were first imaged at the fluorescence microscope (a) and then at the transmission electron microscope after silver enhancement of the nanogold signal (b–p). At the light microscopy level (a) fluorescent labelling is detected at apical areas and at basolateral regions of the P compartment. At the EM level, apical immunogold labelling (c and d shows the area delimited by a red box in b) decorated microvilli membranes (arrows in d), whereas basolateral signal (f–i shows EM images of the area delimited by a blue box in b, while j–p shows details of a different disc) is mainly associated with discrete extents of the plasma membrane (arrows in f,i,j) that frequently coincide with cell-to-cell contacts (arrows in j). Also, immunogold labelling is detected in clusters of EV-like structures (arrowheads in g,i,k,m,n,p), some of which are associated with cell protrusions (insets in f (g) and l (m)). A and P compartments as well as apical (Ap) and basolateral (Bs) regions are indicated in a and b. To help the interpretation, apical and basolateral extracellular spaces are depicted in red (c) and blue (f,i–p), respectively. a to i correspond to the same wing disc, whereas j to p correspond to a different specimen. LD, lipid droplet. Scale bars, 25 μm (a,b), 5 μm (e), 500 nm (c,h,j,l,o), 200 nm (d,f,i,k,m,n) and 100 nm (g,p).