The primary antibodies used in this study were as follows: anti-BRG1 (EPNCIR111A) Abcam Cat# ab110641 (1:10,000); anti-Caspase3 (8G10), Cell Signaling Technology, Cat# 9665 (1:1000); anti-C-Caspase 3, Cell Signaling Technology, Cat# 9661 (1:1000); anti-PARP (46D11), Cell Signaling Technology, Cat# 9532 (1:1000); anti-Ki67 (B56), BD Biosciences, Cat# 550609 (1:500); anti-EpCAM (G8.8), BD Biosciences, Cat# 552370 (1:500); anti-ZO1, Invitrogen, Cat# 40-2200 (1:500); anti-8-OHd G, Abcam, Cat# ab48508 (1:500); anti-LC3A/B (D3U4C), Cell Signaling Technology, Cat# 12741 (1:1000); anti-ATG16L1 (D6A5), Cell Signaling Technology, Cat# 8089 (1:1000); anti-PCNA, Santa Cruz Biotechnology, Cat# SC-7909 (1:1000); anti-P-H3, Cell Signaling Technology, Cat# 9701 (1:1000); anti-E-Cadherin, Cell Signaling Technology, Cat# 3195T (1:1000); anti-DCLK1, Abcam, Cat# ab31704 (1:500); anti-Claudin1, Cell Signaling Technology, Cat# 4933T (1:1000); anti-ChgA, Abcam, Cat# ab715 (1:1000); anti-Hes1, Abcam, Cat# ab71559 (1:500). Raw data of immunoblotting can be found in the source data file.
Anti dclk1
Manufactured by Abcam
Sourced in United Kingdom
Anti-DCLK1 is a protein marker that can be used for the detection and quantification of DCLK1 (Doublecortin-like kinase 1) in various biological samples. DCLK1 is a kinase involved in the regulation of microtubule dynamics and plays a role in neuronal development and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Anti e cadherin, by Cell Signaling Technology (4 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (4 mentions)
Anti gpcr gpr49 lgr5, by Abcam (4 mentions)
Alexa fluor 488 coupled secondary igg, by Thermo Fisher Scientific (4 mentions)
Anti β actin total, by Merck Group (4 mentions)
Anti aire, by Thermo Fisher Scientific (3 mentions)
Anti zo 1, by Thermo Fisher Scientific (2 mentions)
Oct compound, by Sakura Finetek (2 mentions)
Anti ccl21, by Bio-Rad (2 mentions)
Anti ly51, by BioLegend (2 mentions)
Anti krt14, by BioLegend (2 mentions)
Uea 1, by Vector Laboratories (2 mentions)
Tcs sp8 confocal laser scanning microscope, by Leica (2 mentions)
Anti cd4, by BioLegend (2 mentions)
Anti cd8, by BioLegend (2 mentions)
Anti cd44, by Cell Signaling Technology (2 mentions)
Rosettesep human cd45 depletion cocktail, by STEMCELL (2 mentions)
Anti ck19, by Abcam (2 mentions)
Anti epcam, by Abcam (2 mentions)
Anti anxa2, by BD (2 mentions)
16 protocols using anti dclk1
1
Comprehensive Antibody Characterization Protocol
2
Immunofluorescence Imaging of Thymic Tissue
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Paraformaldehyde-fixed frozen tissues embedded in OCT compound (Sakura Finetek) were sliced into 10-μm-thick sections. The sections were stained with anti-CCL21 (Bio-Rad), anti-Aire (eBioscience), anti-Ly51 (Biolegend), anti-Krt14 (Biolegend), anti-DCLK1 (abcam), anti-CD4 (Biolegend), anti-CD8 (Biolegend) antibodies, anti-Foxn1 antiserum50 (link), and UEA-1 (Vector Laboratories). Images were analyzed with a TCS SP8 confocal laser scanning microscope (Leica).
3
Antibody and Drug Protocol for Cancer Research
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Antibodies used in this study were obtained from commercial sources as follows: anti-β-catenin (610153; BD Transduction Laboratories), anti-Axin2 (76G2; Cell Signaling/WB; HPA042344; Sigma/IHC), anti-GFP (sc-9996; Santa Cruz), anti-GAPDH (Epitomics), anti-CD44-APC (C26; BD Pharmigen), APC mouse IgG2b k isotype control (BD Pharmigen), anti-DCLK1 (Abcam) and PE conjugated-goat anti-rabbit IgG H&L (Abcam). EGF, bFGF, and Wnt3a were obtained from R&D systems. Verapamil, Hoechst 33342, PI, KY02111, cisplatin, and oxaliplatin were purchased from Sigma. miR-103/107 precursors and miR-103/107 LNAs (antagomirs) were purchased from Ambion and EXIQON, respectively.
4
Isolation and Characterization of Circulating Tumor Cells
5
Isolation and Analysis of Thymic Epithelial Cells
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For the analysis of thymic epithelial cells (TECs), minced thymuses were digested with 0.5 unit/ml Liberase (Roche) in the presence of 0.02% DNase I (Roche). Single-cell suspensions were stained with antibodies specific for EpCAM (BioLegend), CD45 (eBioscience), Ly51 (Biolegend), Podoplanin (Biolegend), I-Ab (BioLegend), and for the reactivity with UEA-1 (Vector Laboratories). For the analysis of Aire and DCLK1, surface-stained cells were fixed in 5% formaldehyde neutral buffer solution (Nacarai Tesque), permeabilized in 1x permeabilization buffer (eBioscience), and stained with anti-Aire (eBioscience) or anti-DCLK1 (abcam) antibody. For the isolation of TECs, CD45− cells were enriched in magnetic bead conjugated anti-CD45 antibody (Miltenyi Biotec). Multicolor flow cytometry and cell sorting were performed on FACSVerse, FACSAria II, and LSRFortessa (BD Biosciences).
6
Immunofluorescence Analysis of Thymic Microenvironment
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Paraformaldehyde-fixed frozen tissues embedded in OCT compound (Sakura Finetek) were sliced into 10-μm-thick sections. The sections were stained with anti-CCL21 (Bio-Rad, Cat# AAM27, RRID:AB_2072089 ), anti-Aire (eBioscience, Cat# 50593482, RRID:AB_2574257 ), anti-Ly51 (BioLegend, Cat# 108312, RRID:AB_2099613 ), anti-Krt14 (BioLegend, Cat# 905304, RRID:AB_2616896 ), anti-DCLK1 (abcam, Cat# ab31704, RRID:AB_873537 ), anti-CD4 (BioLegend, Cat# 100533, RRID:AB_493372 ), anti-CD8 (BioLegend, Cat# 100701, RRID:AB_ 312740) antibodies, anti-Foxn1 antiserum (Itoi et al., 2007 (link)), and UEA-1 (Vector Laboratories, Cat# B1065, RRID:AB_2336766 ). Images were analyzed with a TCS SP8 confocal laser scanning microscope (Leica).
7
Isolation and Characterization of Circulating Tumor Cells
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Antibodies used include: anti-CD44 (Cell Signaling Technology, Danvers, MA); anti-DCLK1, anti-CD45, anti-EpCAM, anti-CK19 and anti-GPCR GPR49 (Lgr5) (Abcam, Cambridge, MA); anti-AnxA2 (BD Biosciences, Carlsbad, CA), and anti-β-actin (total) (Sigma, St Louis, MO). Anti-PG antibody was generated in our laboratory as described [27 (link)). Alexa Fluor-594 and Alexa Fluor-488 coupled secondary IgG were from Invitrogen (Carlsbad, CA). Three kits from Stem Cell Technologies were used: 1) RosetteSep™ Human CD45 Depletion Cocktail (#1522), 2) RosetteSep™ Human Circulating Epithelial Tumor Cell Enrichment Cocktail (#15127), and 3) EasySep™ Human Whole Blood CD45 Depletion Kit (#18289), (Vancouver, Canada).
8
Immunoblotting Analysis of Cellular Proteins
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Total crypt cellular or nuclear extracts (30–50 μg protein/lane), were subjected to SDS-PAGE and electrotransferred to nitrocellulose membrane. The membranes were blocked with 5% BSA or 5% nonfat dried milk in Tris-buffered saline (TBS) (20 mM Tris-HCl and 137 mM NaCl, pH 7.5) for 1 h at room temperature (21 °C). Immunoantigenicity was detected by incubating the membranes overnight with the appropriate primary antibodies (0.5-1.0 μg/ml in 5% BSA or 5% nonfat dried milk) and Western blot analysis was performed as described [10 (link)]. Antibodies used were anti-DCLK1 (1:1000, ab31704, ab109029) from Abcam, anti-p62/SQSTM1 (1:400, H00008878-M01), anti-LC3B (1:300, NB100-2220) from Novus Biologicals, anti-GAPDH(1:3000), anti-β-actin (1:5000), from Sigma-Aldrich, anti-EZH2 (1:000), anti-H3 (1:1000) were from Cell Signaling Technology.
9
Quantification of Intestinal Tuft Cells
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Small intestines were collected, formalin fixed, and paraffin embedded. Paraffin sections (5 µm) were deparaffinized in xylene and rehydrated in an ethanol gradient. Antigen retrieval was performed in 10 mM sodium citrate buffer, pH 6.0, at 95°C for 20 min. Sections were stained with rabbit polyclonal anti-DCLK1 (1:1,000; Abcam) overnight at 4°C, followed by staining with Alexa Fluor 647–conjugated anti–rabbit secondary antibody (1:4,000; Molecular Probes), and counterstaining with DAPI (1:10,000; Sigma-Aldrich). Images were acquired with the Axio Scan Z.1 Slide Scanner (ZEISS). Quantification was performed blind by counting the total number of tuft cells per crypt-villus with the Zen Blue 2 software (ZEISS).
10
Immunohistochemistry Assay for Stem Cell Markers
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