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Fig. 2. Immunohistochemical localisation of PrP^C and LRP1 on sensory neurons. (A,B) Immunolabelling for cell-surface PrP^C with 2S Alexa-Fluor-594–Fab (red), 3 µm (A) and 9 µm (B) above the plane of the laminin substrate. PrP^C-expressing neurons (1-3; arrowheads point to their surface labelling), plus axons (arrows), and PrP^C-negative substrate cells (visible by their blue DAPI-stained chromatin, asterisks) can be seen in (A); only their cell bodies are visible in B. These images are from a series (supplementary material Fig. S1), collected every 100 nm in vertical steps, which were deconvolved and assembled into ascending stacks of ten sections (i.e. 1 µm optical sections). Neuronal cell bodies shown subsequently in this paper are combined from stacks of 10-30 images taken at a level corresponding to B, with DAPI-stained chromatin. (C,D) Surface immunolabelling of PrP^C (2S Alexa-Fluor-594–Fab, red) and green Alexa-Fluor-488-labelled anti-LRP1 (C) or Ctx (D) on sensory neurons fixed at 37°C before immunolabelling. On the cells shown, 37% and 77% of PrP colocalised with LRP1 and Ctx (data are in supplementary material Tables S1a and S1b). (E) Neuron with surface PrP^C prelabelled at 10°C with Alexa-Fluor-594–Fab (red), then allowed to endocytose at 37°C for 1 minute, then fixed, permeabilised and immunolabelled for LRP1. It had endocytosed 82% of its labelled PrP^C, most to perinuclear tubular structures (yellow owing to colocalised LRP1); arrow points to labelled PrP^C still on the surface. (F) Transmission EM showing labelling within a coated pit on a neuron that has endocytosed (1.5 minutes at 37°C) its prelabelled 5 nm gold-Fab to PrP^C, and 10 nm gold-₂M^*. Bars, 5 µm (A,C-E), 100 nm (F).

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