Fig. 1

Compartmental localization of CD63-GFP⁺ ILVs in CD63-GFP^f/+ neurons. a Schematic diagram of the gene targeting strategy to insert the CD63-copGFP-6xHis cassette into the Rosa26 locus, in the intron between endogenous exons 1 and 2, for the generation of exosome reporter CD63-GFP^f/f mice. copGFP: GFP cloned from copepod Pontellina plumata; h or mCD63: human or mouse CD63; b Representative images of the distribution of CD63-GFP⁺ puncta in specific compartments of cultured primary neurons. Subpanels i: βIII-tubulin staining; ii: CD63-GFP fluorescence; iii: MAP2 and DAPI staining; vi: merge of CD63-GFP fluorescence and MAP2 staining; vii: magnified view of βIII-tubulin staining. Yellow arrows (subpanels iv and v): axons (βIII-tubulin⁺MAP2⁻) with sparse overlap of CD63-GFP⁺ puncta; White arrows (subpanels iv and viii): dendrites (βIII-tubulin⁺MAP2⁺) with abundant CD63-GFP⁺ puncta; Gray arrows (subpanels iv and viii): CD63-GFP⁺ puncta in the soma; Scale bar: 50 μm; v–viii, magnified confocal images of axons and dendrites taken with the 63x objective lens; c Quantification of CD63-GFP intensity in the soma, dendrites (βIII-tubulin⁺ MAP2⁺), and axons (βIII-tubulin⁺ MAP2⁻) of cultured primary neurons. n = 40–41 neurons/group. P-value was calculated using one-way ANOVA followed by a Tukey post-hoc test. d Representative images of CD63-GFP with endogenous mouse CD63 immunoreactivity. Scale bar: 10 μm; An anti-CD63 antibody specifically recognizing mouse CD63 was used. The magnified view of the CD63-GFP⁺ neuron (subpanel i) with each channel (as indicated near the image) is shown in ii–vi, respectively. white arrows: overlapped CD63-GFP with endogenous CD63 immunoreactivity