Mucin biosynthesis and cell proliferation of distal colon.

<p>A) GalNAz-incorporation during mucin biosynthesis was analyzed every hour for 12 h and at 24 h. Samples from distal colon were fixed after 1 to 24 h, as indicated, and GalNAz was detected by TAMRA (red) in combination with immunostaining of Muc2 (green). GalNAz is first identified in the Golgi region (1 h) and all cells in the crypt bottom are intensely stained during the first 7 h whereafter the staining decreases. Mucus filled goblet cells in the mid to upper crypt are stained green and GalNAz incorporation in the mucin vesicles can be observed from 5 to 10 h of labeling (arrows). The basal membrane and lamina propria are stained after 3 h with highest intensity after 11–12 h and the muscular layers are stained red after 7 h. Tissue not injected with GalNAz but stained with TAMRA was used as a negative control (Neg). Scale bars are 50 µm. B) Low magnification pictures of colon labeled with GalNAz (stained red by TAMRA) for 3, 7 and 11 h. Scale bars are 100 µm. C) Western blot of scraped mucus from mouse colon with GalNAz-labeled mucins separated by agarose-acrylamide electrophoresis. GalNAz was reacted with alkyne-biotin and reduced prior to separation on gel. The blot was detected with streptavidin (SA) or anti-MUC2N3/C3 (Muc2) and the glycosylated Muc2 band is biotinylated demonstrating GalNAz-incorporation into Muc2. D) BrdU was injected simultaneously with GalNAz and was detected by immunostaining (red) after 1, 6 and 12 h. Scale bars are 50 µm.