Table 1.

Quantitation of Antibody Colocalization in Confocal Immunofluorescence Images

Antibodies ComparedPercentage of Colocalization (Mean ± sd)n
VSR antibodies versus AtSYP21 versus 14G7 antibodies (Figure 2)
    VSR:CT and VSR:14G794 ± 612
    VSR:AtSYP21 and CT:AtSYP2191 ± 615
YFP reporters versus anti-GFP in transgenic BY-2 cells (Figure 3)
    GONST1-YFP:anti-GFP93 ± 623
    YFP-BP-80:anti-GFP96 ± 318
YFP reporters versus anti-VSR in transgenic BY-2 cells (Figure 5, panels 1 and 2)
    YFP-BP-80:anti-VSR97 ± 39
    GONST1-YFP:anti-VSR8 ± 215
YFP reporters versus AtSYP21 in transgenic BY-2 cells (Figure 5, panels 3 and 4)
    YFP-BP-80:anti-AtSYP2183 ± 26
    GONST1-YFP:anti-AtSYP212 ± 311
YFP reporters versus anti-ManI in transgenic BY-2 cells (Figure 5, panels 5 and 6)
    YFP-BP-80:anti-ManI4 ± 310
    GONST1-YFP:anti-ManI91 ± 414
YFP reporters versus anti-VSR in transgenic BY-2 cells treated with wortmannin (33 μM) for 3 h (Figure 8C)
YFP-BP-80:anti-VSR85 ± 510
YFP reporters versus FM4-64–labeled PVCs (Figure 14)
    YFP-BP-80:FM4-6485 ± 419
    GONST1-YFP:FM4-6411 ± 425
  • YFP reporters in transgenic BY-2 cells were studied by confocal immunofluorescence localization. Antibody marker for YFP was anti-GFP; antibody marker for Golgi was anti-ManI; and antibody marker for VSR protein and PVC was anti-VSR. FM4-64 is a fluorescent dye that labels prevacuolarment after endocytosis. Quantitation of the extent of colocalization for the two antibodies (or proteins) was performed from one direction only (i.e., to determine how much of the YFP-BP-80 reporter proteins colocalized with anti-VSR and not the other way around) as described previously (Jiang and Rogers, 1998). Percentage of colocalization is expressed as the mean ± sd for the number of cells analyzed (n). Also, a t test was performed to compare results from the individual paired assays of the two reporters expressing in BY-2 cells. The resulting P values for the comparison were statistically significant (P < 0.01).