Paraffin-embedded sections were dewaxed and rehydrated. The sections were then immersed in Tris/EDTA (pH 9.0) and incubated for 15 min at 98 °C for antigen retrieval. The sections were incubated with serum for 30 min at room temperature to block non-specific antigen-binding sites. Anti-Ki67 antibody, anti-NF-κB p65 antibody, anti-Lgr5 antibody (Abcam, Cambridge, MA, USA), anti-lysozyme antibody, and anti-Villin antibody (Proteintech, Chicago, IL, USA) was diluted as recommended by the manufacturer, added to the sections, and incubated overnight at 4 °C. Unbound antibodies were washed away with PBS. Sections were incubated in 0.3% H2O2 for 15 min to block endogenous peroxidase. Secondary antibody was added and incubated at 37 °C for 2 h. DAB was added to detect positive cells.
Anti lgr5 antibody
Manufactured by Abcam
Sourced in United States
The Anti-Lgr5 antibody is a protein-based reagent that specifically binds to the Lgr5 protein, which is a marker for intestinal stem cells. It can be used in various laboratory techniques, such as immunohistochemistry, to detect and study the Lgr5-positive cells in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Dab kit, by Merck Group (3 mentions)
Anti lysozyme, by Abcam (3 mentions)
Anti villi, by Abcam (3 mentions)
Anti ki67 antibody, by Abcam (1 mentions)
Anti nf κb p65 antibody, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Anti gapdh antibody, by ABclonal (1 mentions)
Anti β catenin, by ABclonal (1 mentions)
Anti β tubulin, by ABclonal (1 mentions)
Ecl chemiluminescence detection kit, by Vazyme (1 mentions)
Anti olfm4 antibody, by Cell Signaling Technology (1 mentions)
Anti ki67 antibody, by Novus Biologicals (1 mentions)
Enhanced chemiluminescent substrate, by Merck Group (1 mentions)
Anti tph1 antibody, by Abcam (1 mentions)
Anti gdnf antibody, by Abcam (1 mentions)
Anti β catenin antibody, by Proteintech (1 mentions)
6 protocols using anti lgr5 antibody
1
Immunohistochemistry of Intestinal Markers
2
Immunohistochemical Analysis of Intestinal Lgr5, Ki67, Lysozyme, and Villi
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The 4 μm-thick sections of paraffin-embedded small intestine sections were dewaxed and rehydrated with citrate buffer. Then, the sections were boiled in 10 mM/L citrate buffer solution (pH 9.0) for antigen retrieval according to the standard procedures. After antigen retrieval, the sections were incubated with serum for 1 h at room temperature to block nonspecific antigen-binding sites and then with anti-Lgr5 antibody (1 : 50 dilution, Abcam, Cambridge, MA, USA), anti-Ki67 antibody (1 : 300 dilution, Novus a biotechne brand), anti-lysozyme (1 : 800 dilution, Abcam, Cambridge, MA, USA), or anti-villi (1 : 800 dilution, Abcam, Cambridge, MA, USA) overnight at 4°C. Sections were then incubated in secondary antibody for 30 min at 37°C. Positive cells were detected using DAB kit (Sigma-Aldrich). The images were captured, and positive staining was quantified objectively by the IPP software as described previously in a blinded fashion.
3
Quantification of Intestinal Stem Cells and Proliferation
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Mice were killed at 3.5 days WBI, and the small intestine was fixed with neutral formalin. Then the tissues were dehydrated and embedded with paraffin. Then the sections were boiled in 10 mM/L citrate buffer solution (pH 9.0) for antigen retrieval according to the standard procedures. After antigen retrieval, the sections were incubated with serum for 1 h at room temperature to block non-specific antigen-binding sites, and then with anti-Lgr5 antibody (1:50 dilution, Abcam, Cambridge, MA, USA), anti-Ki67 antibody (1:300 dilution, Novus a biotechne brand), anti-lysozyme (1:800 dilution, Abcam, Cambridge, MA, USA) or anti-villi (1:800 dilution, Abcam, Cambridge, MA, USA) overnight at 4 °C. Sections were then incubated in secondary antibody for 30 min at 37 °C. Positive cells were detected using DAB kit (Sigma Aldrich). The images were captured and positive staining was quantified objectively by the IPP software as described previously in a blinded fashion.
4
Western Blotting of Intestinal Proteins
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Total proteins from tissues and organoids were harvested using RIPA Lysis Buffer (Beyotime) with phenylmethyl sulfonyl fluoride (PMSF) protease inhibitor and phosphatase inhibitor. Samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes, followed by blocking with 5% skimmed milk for 1 h. The membranes were then incubated with primary antibodies and the corresponding secondary antibodies (all 1:1000 dilutions): anti-Lgr5 antibody (Abcam, ab75850), anti-Olfm4 antibody (Cell Signaling, 39141), anti-GAPDH antibody (Abclonal, AC001), anti-GPR41 antibody (Abclonal, A12636), anti-GPR43 antibody (Proteintech, 19952-1-AP), anti-β-tubulin (Abclonal, AC015), anti-Axin2 antibody (Proteintech, 20540-1-AP), anti-β-catenin (Abclonal, A19657), anti-Wnt3 antibody (Abclonal, A9328), and HRP conjugated Goat anti-rabbit IgG (Antgene, 72-8067). The bands were detected using a chemiluminescence imaging system (UVP) with ECL chemiluminescence detection kit (Vazyme). The relative fold change in protein expression was normalized to that of GAPDH or β-tubulin.
5
Immunohistochemical Analysis of Intestinal Stem Cells
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The 4‐μm‐thick sections of paraffin‐embedded small intestine sections were dewaxed and rehydrated with citrate buffer. Then, the sections were boiled in 10 mM/L citrate buffer solution (pH 9.0) for antigen retrieval according to standard procedures. After antigen retrieval, the sections were incubated with serum for 1 hour at room temperature to block non‐specific antigen‐binding sites; then, the sections were incubated with anti‐Lgr5 antibody (1:50 dilution; Abcam, Cambridge, MA), anti‐Ki67 antibody (1:300 dilution; Novus, Littleton, CO), anti‐lysozyme (1:800 dilution; Abcam) or anti‐villi (1:800 dilution; Abcam) overnight at 4°C. Sections were then incubated in secondary antibody for 30 minutes at 37°C. Positive cells were detected using a DAB kit (Sigma Aldrich, St. Louis, MO). The images were captured, and positive staining was quantified objectively by the integrated performance primitives (IPP) software as described previously in a blinded fashion.
6
Immunoblotting Analysis of Proteins
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Total proteins were extracted and quantified. Protein was separated by 10% SDS‐PAGE and transferred onto a PVDF membrane. The membrane was incubated with primary antibody at 4°C overnight, then incubated with the secondary antibody for 1 hour at room temperature. The immunoblots were detected by an enhanced chemiluminescent substrate (Millipore). Antibodies: anti‐RET antibody (1:1000; Abcam), anti‐Lgr5 antibody (1:1000; Abcam), anti‐β‐catenin antibody (1:1000; Proteintech, USA), anti‐TPH1 antibody (1:500; Abcam), anti‐GDNF antibody (1:500; Abcam), anti‐Ngn3 antibody (1:300; Abcam), anti‐β‐actin antibody (1:1000; Zhongshan Gold Bridge, Beijing, China), goat anti‐mouse IgG (1:1000; Zhongshan Gold Bridge) and goat anti‐rabbit IgG (1:1000; Zhongshan Gold Bridge).
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