Specimens were harvested and fixed overnight in 10% formalin/phosphate-buffered saline and embedded in paraffin blocks. The proliferative capacity of the small intestine and apoptosis were determined by immunohistochemistry for BrdU (Abcam, ab6326), Ki67 (Abcam, ab15580) and active caspase-3 (Cell Signaling Technologies, 9579S). The cellular composition of the intestine was determined using antibodies for Dclk1 (Abcam, ab37994), chromogranin A (Abcam, ab15160), Muc2 (Santa Cruz Biotech, sc-15334), lysozyme (Dako, A0099), and synaptophysin (Abcam ab32127). Biotinylated secondary antibodies were applied at 1:1000 dilutions and visualized by using diaminobenzidine peroxidase (DAB) substrate (Invitrogen, Carlsbad, CA). Slides were counterstained with hematoxylin. For immunofluorescent staining, secondary antibodies were applied at 1:500 dilution and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).
Ab37994
Manufactured by Abcam
Sourced in United Kingdom
Ab37994 is a purified recombinant protein product from Abcam. It is designed for use in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
36 e cadherin, by BD (2 mentions)
Mouse anti e cadherin, by BD (2 mentions)
Diaminobenzidine peroxidase dab substrate, by Thermo Fisher Scientific (1 mentions)
Ab32127, by Abcam (1 mentions)
A0099, by Abcam (1 mentions)
Ab15580, by Cell Signaling Technology (1 mentions)
Ab6326, by Abcam (1 mentions)
Hct116, by American Type Culture Collection (1 mentions)
Ab134188, by Abcam (1 mentions)
5 protocols using ab37994
1
Intestinal Cellular Composition and Proliferation
2
Comparative Analysis of Colon Cell Lines
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NCM460 and HCT116 cell lines were purchased from American Type Culture Collection. NCM460 cells are a type of normal colon cell, and HCT116 is a human colon cancer cell line. Both cell types were maintained in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. We used a polyclonal rabbit anti-DCLK1 antibody (ab37994; Abcam, Cambridge, UK) and a rabbit anti-ALDH1 mAb (ab134188; Abcam) as positive controls.
3
Histological and Immunofluorescence Analysis of Intestinal Tissue
4
Histological and Immunofluorescence Analysis of Intestinal Tissue
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The small intestine was removed and divided into proximal and distal sections before overnight fixation in 4% paraformaldehyde. The tissue was then embedded in paraffin and cut into 5-μm thick sections. For both histology and immunofluorescence, sections were initially deparaffinized and rehydrated. Goblet cells were identified by Alcian blue/nuclear red staining and enumerated along the crypt-villus axis by quantitative microscopy. For immunofluorescence, heat-mediated Ag retrieval was performed in Tris-EDTA buffer 0.05% Tween-20 (pH 9) for 20 min. Afterwards, the slides were washed in PBS and blocked in PBS containing 3% BSA, 3% donkey serum, 0.1% Triton X-100, and 0.1% saponin for 1 h at room temperature. Primary Abs were incubated overnight at 4°C and secondary Abs were applied for 1.5 h at room temperature. Primary Abs included the following: rabbit anti-DCLK1 (1:250 dilution, ab37994; Abcam) and mouse anti–E-cadherin (1:400 dilution, 36/E-cadherin; BD Biosciences). DNA was labeled with DAPI (0.5 μg/ml).
5
Stem Cell Marker Expression Analysis
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The following primary antibodies were used with the protocol described in Supplemental Experimental Procedures : rabbit polyclonal anti-human DCLK1, rabbit monoclonal anti-human BMI1 (#ab37994, #ab126783; Abcam, Cambridge, MA), mouse monoclonal anti-human CD44, rabbit monoclonal anti-human Cyclin D1, c-Jun, Wnt3a, Wnt5a, DVL2, DVL3, GSK3β, phosphorylated GSK3β, TCF1/TCF7, β-catenin, GAPDH (#3570, #2978, #9165, #2721, #2530, #3224, #3218, #12456, #5558, #2203, #8480, #5174; Cell Signaling Technology, Beverly, MA) and mouse anti-human c-Myc (sc-40, Santa Cruz Biotechnology).
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