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Fig. 6. FN at the base of clefts translocates into the bud whereas newer FN assembles behind older FN. (A) E12 SMGs were labeled with Alexa Fluor-647-FN-containing media for 6 hours and imaged by time-lapse confocal microscopy. After 1 hour 48 minutes of imaging, medium was removed and replaced with fresh medium, and imaging was continued (supplementary material Movie 7). The initiation points of two clefts are labeled with open arrowheads. The bases or bottoms of each of these clefts move inward into the interior of the gland as indicated by filled arrowheads. (B) To image FN displacement, SMGs were incubated with Alexa Fluor-488-FN (green) for 12 hours, washed, and then treated with Alexa Fluor-647-FN (red) for two additional hours. The earliest FN (green) was concentrated at the bottom and lower sides of the cleft, whereas the FN added later (red) assembled in the bud basement membrane and the upper portion of the clefts. (C) Higher-magnification images of clefts at 16 hours from time-lapse images in (A) showing concentration of FN in a wedge-like pattern. Bars, 100 µm.
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