Ocedure (i.e., dissection followed by immersion into a fixative answer without any prior fixation) the proteins tended either to leak in to the lamina propria (galectin-4, Fig. 3a) or to form cellular aggregates (galectin-6, Fig. 3b). Even so, even when tissues were fixed by intracardiac perfusion (e.g., Figs 3c ), some variability inside the retention of cytosolic galectin-4 and -6 was observed (compare galectin-4 in Fig. 3c and galectin-6 in Fig. 3d with Figs 2k and 2l). Such a loss of cytosolic galectin-4 at fixation had already been noted in human T84 cells (Huflejt et al. 1997) and could also be observed in studies by others in mice and pigs (Chiu et al. 1992; Nio-Kobayashi et al. 2009). This fainter cytosolic galectin-4 and -6 staining helped to reveal the presence of those proteins in otherwise unnoticed locations. Endosomes in enterocytes (arrowheads in Fig. 3a and information not shown). A weaker galectin-4 cytosolic signal revealed subapical granules within the cytoplasm of enterocytes, the tall columnar cells which are responsible for the final digestion and absorption of nutrients, electrolytes, and water. In cell cultures, BFH772 web Stechly and colleagues (Stechly et al. 2009) have shown the granules to become early and apical recycling endosomes but not late endosomes or lysosomes. They alsoGalectin-4/-6 Expression within the Digestive TractFigure 4. Galectin-4 and -6 expression within the colon of dextran sodium sulfate (DSS)-treated mice. Expression of galectin-4 (two left columns) or galectin-6 (appropriate column) in the C57BL/6J (left column) or 129/Sv (two correct columns) background.The arrows point toward galectin-4 decorated cells in the lamina propria. The insets in 4c and 4e are enlargements with the region to which the arrows point. The inset in 4k is often a image from the crypt shown in 4k, using a shorter exposure time to be able to reveal the galectin-6-positive granules inside the crypt. The labels d1 (day 1) to d4 (day 4) around the left represent the period throughout which DSS was in the drinking water. The anti-galectin-4 or -6 staining is shown in red, anti-Muc2 in green, and DAPI-stained nuclei in blue. Scale bars = 40 . The same settings and exposure occasions had been made use of for all photos, except for the inset in 4k.demonstrated that galectin-4 is endocytosed then recycled back for the apical surface with the cells and how this endocytic-recycling pathway of galectin-4 is required for the apical trafficking of glycoproteins (Stechly et al. 2009). In contrast, galectin-6 was never noticed in these endosomes and, thus, in spite with the sequence similarities it PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/2012433 shares with galectin-4, it is actually unlikely to be part of the endocyticrecycling pathway.Cell membranes (Figs 3c, 3d, too as inset in Fig. 3d displaying an enlargement of an additional villus). The part played by galectin-6 in mouse enteroendocrine cell physiology may deserve additional investigation. Goblet cells (Figs 2k, 3a, 3c). The galectin-4 and -6 proteins have been also detected within the cytoplasm of goblet cells, the mucussecreting cells within the digestive tract. The signal appeared typically stronger than within the neighboring enterocytes (e.g., Fig. 3a, arrow). The expression of galectin-4 and -6 within the goblet cells seemed even more sensitive to the tension induced by dissection than elsewhere inside the epithelium, as their subcellular localization was more variable in these cells than in any other cell type. On numerous occasions, galectin-4 appeared concentrated in the base on the huge mucus vesicle (inset in Fig. 2k, arrow in Fig. 3a.