Fig. 1

Recommendations for the identification of GVBs in a research setting: three criteria for the validation of GVB identity in tissue and cell models. Criterion 1: Immunoreactivity for the common GVB marker CK1δ in the core (tissue a, cells b). Criterion 2: Immunoreactivity of the CK1δ-positive core for another common GVB core marker determined by double immunolabeling (tissue c, cells d). Alternatively, staining of adjacent sections may be performed in experiments on tissue. Additional common GVB core markers are pPERK, peIF2α, pIRE1α, CK1ε, CHMP2B and pTDP-43. Note that CHMP2B does not exclusively stain GVBs in cultured cells and that pTDP-43 has not been tested in the in vitro model. Criterion 3: Visualization of characteristic GVB morphology, including the presence of the GVB membrane and/or vacuole (tissue e, cells f). The GVB membrane is preferably visualized by immunodetection of the lysosomal membrane marker LAMP1 (f) or LIMP2. In tissue, GVB morphology may also be visualized employing the routine H&E staining that is used for diagnostics. Alternatively, chromogenic peroxidase-catalyzed immunodetection of a GVB core marker often reveals GVB core, vacuole and membrane (e). These criteria are also of use for the validation of novel GVB markers that can be investigated by examining their co-localization with CK1δ (criterion 1) and an additional common GVB marker (criterion 2) as well as their subcellular localization to GVB core/membrane (criterion 3). a, c and e show immunolabeling of human AD hippocampus. b, d and f show immunolabeling of cultured primary mouse neurons with seeded tau pathology [143]. Cell nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI) in a-d and with hematoxylin in e. In a-d and f immunofluorescence and in e immunohistochemistry using the chromogen 3,3′-diaminobenzidine (DAB) was performed. Arrowheads in f point to GVBs. See text for details