Fig. 1. Stimulation-dependent surface labeling and internalization of non-secreted PRL. (A-D) Lactotrophs were stimulated for 5 min at room temperature in high [K⁺] external solution containing anti-PRL antibodies, then rinsed, and fixed at the indicated time point, permeablized and processed for indirect immunofluorescence. (A) t=0; (B) t=10; (C) t=20; (D) t=80 minutes. (E,F) Surface labeling of non-internalized anti-PRL. Cells were labeled with anti-PRL as above and then fixed and processed for indirect immunofluorescence without permeablization of the plasma membrane. (E) t=0; (F) t=10 minutes. Deconvolved images from the middle of a z-series are shown. DAPI staining shown in blue. Scale bar is 10 µm for all fluorescence images. (G) Gold-labeled secondary antibody was applied to non-permeablized cells fixed at t=0 to reveal anti-PRL in surface-exposed dense cores. Bar, 400 nm.