single-plex rnascope probes against mouse lgr5  (Advanced Cell Diagnostics Inc)

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: ATF2 and ATF7 Are Critical Mediators of Intestinal Epithelial Repair

doi: 10.1016/j.jcmgh.2020.01.005

Figure Lengend Snippet: ATF2 and ATF7 are required to prevent apoptosis and sustain goblet cell differentiation after DSS-induced damage. The experiment was performed as described in Figure 4 A . ( A ) Representative images of immunohistochemistry for BrdU in colons of DSS-treated mutant and control animals. Scale bar : 750 μm. ( B ) Quantification of BrdU-positive cells in intact colonic crypts. ( C ) qRT-PCR of the isolated epithelial fraction for epithelial stem cell markers Cd44 , Lgr5 , and Ascl2 . ( D ) Representative images of colonic RNA scope in situ hybridization for Lgr5 messenger RNA in homeostasis (as described in Figure 1 A ) and on exposure to 2% DSS. ( E ) quantification of Lgr5 messenger RNA particles per colonic Lgr5-positive stem cell ( left panel ) and quantification of Lgr5 -positive cells per colonic crypt ( right panel ). Mice were analyzed for homeostasis: Atf2 wt/wt Atf7 +/+ (n = 8) and Atf2 -/- Atf7 ko/ko (n = 8), and for 2% DSS: Atf2 wt/wt Atf7 +/+ (n = 4) and Atf2 -/- Atf7 ko/ko (n = 4). ( F ) Representative immunohistochemistry image of colonic periodic acid–Schiff (PAS) staining. ( G ) Relative abundance of PAS-positive cells per crypt epithelial cells. ( H ) qRT-PCR for secretory epithelial markers in isolated colonic epithelial fraction. ( I ) TUNEL staining (green) with 4′,6-diamidino-2-phenylindole counterstain (blue). Upper panels : colons from mice not treated with DSS (n = 5 per group). Lower panels : colons from mice treated with DSS for 7 days (n = 6 mice per group). Scale bar : 600 μm. ( J ) Quantification of TUNEL-positive cells per microscopic field (5 random fields per mouse). Mice were analyzed for homeostasis: Atf2 wt/wt Atf7 +/+ (n = 5) and Atf2 -/- Atf7 ko/ko (n = 5), and for 2% DSS: Atf2 wt/wt Atf7 +/+ (n = 6) and Atf2 -/- Atf7 ko/ko (n = 6). ( B , C , G , and H ) n = 12 mice per group. Graph bars show means and SEM. * P < .05, ** P < .01, *** P < .001, Student t test for panels B , C , G , and H , 1-way analysis of variance test for panels E and J followed by the Bonferroni test for selected groups. Rel., relative.

Article Snippet: The single-plex RNAscope probes against mouse Lgr5 (312171; Advanced Cell Diagnostics) were used.

Techniques: Cell Differentiation, Immunohistochemistry, Mutagenesis, Control, Quantitative RT-PCR, Isolation, RNAscope, In Situ Hybridization, Staining, TUNEL Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: ATF2 and ATF7 Are Critical Mediators of Intestinal Epithelial Repair

doi: 10.1016/j.jcmgh.2020.01.005

Figure Lengend Snippet: ATF2 and ATF7 are required to protect epithelial cells against TNF-induced death in organoid culture. Colonic epithelium was isolated from Atf2 wt/wt Atf7 +/+ and Atf2 -/- Atf7 ko/ko mice and cultured as organoids (n = 3, independent organoid cultures). ( A ) qRT-PCR for the floxed region of Atf2 and Atf7 in colonic organoids. ( B ) Representative brightfield images of colonic organoids 72 hours after reseeding. ( C ) Quantification of organoid area and perimeter. ( D ) EdU incorporation assay using flow cytometry 72 hours after reseeding single cells (n = 3 for 2 independent experiments). ( E ) qRT-PCR for stem cell markers Lgr5 and Olfm4 . ( F ) PI viability assay on colon organoids challenged with TNF-α for 12 hours. Scale bar : 250 μm. Red fluorescence indicates cell death. ( G ) Percentage of nonviable organoids quantified per well (n = 2). Graph bars show means and SEM, ∗∗ P < .01, ∗∗∗ P < .001, Student t test.

Article Snippet: The single-plex RNAscope probes against mouse Lgr5 (312171; Advanced Cell Diagnostics) were used.

Techniques: Isolation, Cell Culture, Quantitative RT-PCR, Flow Cytometry, Viability Assay, Fluorescence

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: ATF2 and ATF7 Are Critical Mediators of Intestinal Epithelial Repair

doi: 10.1016/j.jcmgh.2020.01.005

Figure Lengend Snippet: Primers Used for qRT-PCR Experiments

Article Snippet: The single-plex RNAscope probes against mouse Lgr5 (312171; Advanced Cell Diagnostics) were used.

Techniques: