Core Abs stain strings only after denaturation: strings contain PrPSc but no detectable PrPC. (A) Fixed ScGT1 costained with N-terminal mAb 8B4 (red) and with core mAb EP1802Y (green). In c and d, cells were denatured with FA before staining. After FA, EP1802Y largely codecorates strings (circles; arrows, short strings) outlined by 8B4 (compare c with d); there was no detectable EP1802Y staining of strings in nondenatured cells (a and b). (B) Pairs of N- and core Abs used to costain strings in this study. Core Abs stained strings only after denaturation, whereas N-Abs decorated them natively. Nevertheless, denaturation still increased the staining intensity of 8B4 and SAF32 (see Fig. S3 F). The dependence of epitopes on denaturation is roughly indicated by their print size on top of the PrP map. (C) In growth-arrested ScGT1, webs costain with SAF32 and core 8H4 after FA denaturation. Variable relative red and green intensities indicate varied proportions of FL PrPSc/PrP27-30 along strings. Images were 3D deconvolved. (D) ScGT1 were costained with SAF32 (a) and with 8H4 (b) after FA denaturation. Two strings codecorated by both Abs (d, orange) are bridged by a PrP27-30 strand (circled; d, red). (E and F) PrPSc strings do not coincide with cytoplasmic PrP27-30 deposits. ScGT1 were costained with SAF32 and core 8H4 using FA denaturation. z-stacks were acquired (z = 0 is dorsal). (E) 3D-deconvolved images are shown for three planes a total of 5.4 µm apart (colors superimposed). Notice that strings stain in orange and are concentrated in the dorsal plane (a and b). In contrast, abundant cytoplasmic PrP27-30 stains exclusively in green (b and c). (F) A detail of the cell in E. The scheme depicts strings staining with both Abs (red and green) near the top of the cell (z = 0 µm) and intracellular PrP27-30 (green; z = 4.6 µm), which is negative for N-terminal staining. (G) PrPSc strings are codecorated with 8B4 and EP1802Y in Min6/RML but are absent from uninfected cells (FA denaturation protocol).
