Fig 1: Characterization by immunofluorescence of CSC. (A) Representative images of cell nuclei stained with DAPI (blue) in CRAC. Immunostaining with (B) CD44+ marker coupled to PE-Cy7 (red signal) and (C) CD24+ marker coupled to FITC (green signal) in CRAC. Scale bar, 5.0 µm. Representative images of (D) cell nuclei with DAPI and immunostaining with (E) CD44+ marker coupled to PE-Cy7 (with red signal) and (F) CD24+ marker coupled to FITC (green signal) in one CSC isolated from CRAC tumors. Scale bar, 4.8 µm. Representative image of differentiated CSCs to epithelial cells with (G) EGFR+ marker, (H) CD17+ marker and (I) CK20+ marker. Scale bar, 5.0 µm. Arrows indicate cells with positive signals. These were observed with an epifluorescence microscope. CSC, cancer stem cells; CRAC, colorectal adenocarcinoma; EGFR, epidermal growth factor receptor.
Fig 2: Culture of mouse small intestinal organoids in decellularized extracellular matrix (dECM)‐based bioinks and Matrigel. (a) Formed intestinal organoids per field of view at 100× at Day 7 of three consecutive passages. Mean ± S.D. (n = 16 from four organoids cultures). One‐way ANOVA. *p < 0.05 and **p < 0.01. (b) Analysis of organoids diameters at Day 7 of three consecutive passages. Mean ± S.D. (n = 80 from four organoids cultures). One‐way ANOVA. ****p < 0.0001. (c) Typical bright field images of formed organoids at Days 1, 3, 5, and 7 at first passage within bioinks and Matrigel. Scale bar 100 μm. (d) Collective ratio of formed enterocysts to cell aggregates at Day 7 of Passage 1. Mean ± S.D. (n = 7 organoids cultures). One‐way ANOVA. ****p < 0.0001. (e) Typical bright field image and immunofluorescence analysis of an enterocyst with Matrigel. Showing villi enterocyte marker CK‐20 and proliferation marker Ki‐67. Scale bar 100 μm
Fig 3: Differentiation pattern of intestinal organoids in decellularized extracellular matrix (dECM-HA-ink II) and Matrigel. (a) Whole-mount immunofluorescence of cultured organoids at Day 7 of Passage 1. Showing villi enterocyte marker CK-20, goblet cell marker mucin-2 (MUC-2), enteroendocrine cell marker chromogranin (CHGA), Paneth cell marker lysozyme (LYZ) and proliferation marker Ki-67. Scale bar 100 µm. (b) Ratio of differentiated marker positive cells to other cells per organoid. Mean ± S.D. (n = 10 organoids). Two-sided t-test. *p < 0.05, ***p < 0.001, and ****p < 0.0001
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