Figure 4. Whole-mount Immunolocalization analysis of HA₃-TTN5 in Arabidopsis including colocalization with Golgi marker and localization in BFA bodies.

(A-B), Representative images showing whole-mount immunostaining of HA₃-TTN5 seedlings with different types of markers for colocalization analysis. (A), Detection of HA₃-TTN5 (chicken α-HA primary antibody, Alexa 555-labeled secondary α-chicken antibody) with Golgi and TGN marker ARF1 (rabbit α-ARF1 primary antibody, Alexa-488-labeled secondary α-rabbit antibody). Both fluorescence signals were detected in vesicle-like structures in root cells in close proximity to each other but mostly not colocalizing. The experiment was repeated twice with three seedlings. (B) Detection of HA₃-TTN5 (rabbit α-HA primary antibody, Alexa-488-labeled secondary α-rabbit antibody) and staining with lipid membrane dye FM4–64 after brefeldin A (BFA) treatment (72 μM, 1 h). Alexa-488 signals colocalized with FM4–64 in BFA bodies in root cells. The experiment was repeated three times with three seedlings. (C), in comparison, YFP fluorescence in YFP-TTN5 seedlings, co-analyzed with FM4–64 after BFA treatment (36 μM, 30 min). YFP fluorescence signals colocalized with FM4–64 in BFA bodies similar as in (B). The experiment was performed once with three independent YFP-TTN5 lines.

Colocalizing signals in the two channels are indicated by filled white arrowheads, whereas signals that do not colocalize in the two channels are indicated by empty white arrowheads. Scale bar overview: 50 μm, close-up: 10 μm.