Human Dkk-3 Antibody

Detection of Dkk‑3 in Human Brain Hippocampus. Dkk‑3 was detected in immersion fixed paraffin-embedded sections of Human Brain Hippocampus using Goat Anti-Human Dkk‑3 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1118) at 3 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC004). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic (Catalog # VCTS021). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm in neurons. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Detection of Human Dkk-3 by Immunohistochemistry Loss of DKK3 protein expression in human breast cancer.(A) Normal mammary epithelial cells showing moderate, predominantly cytoplasmic, DKK3 immunoreactivity whereas (B) primary antibody negative control is free of signal. (C-E) Weak DKK3 protein expression is observed in breast tumor samples, with lowest intensity in IHC-defined (C) TNBC cases compared to (D) HER2-positive and (E) luminal carcinomas (representative images). (F) Box plot analysis demonstrating a significant down-regulation of DKK3 in tumors of the TNBC (n = 54), the HER2-positive (n = 47) and luminal subtype (n = 362) compared to normal breast tissues (n = 11). Horizontal lines: grouped medians. Boxes: 25-75% quartiles. Vertical lines: range, minimum and maximum. * P < 0.05, *** P < 0.001, IRS: immunoreactive score. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/27467270), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Dkk-3 by Western Blot Ectopic expression of DKK3 in MB231 cells disrupted Wnt signalling. (A) Subcellular localization of beta -catenin and active-beta -catenin by immunofluorescence staining. (B) Western blot analysis of beta -catenin, its downstream targets, JNK and EMT markers. (C) RT-PCR analysis of representative stem cell markers in DKK3-transfected MB231 cells. *Indicates significantly down-regulated bands. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/23890219), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Dkk-3 by Immunocytochemistry/ Immunofluorescence Overview of Multi-dimensional Microscopic Molecular Profiling (MMMP).The overall MMMP approach is depicted using an example tissue section from normal human duodenum (sample #1.9.7). (a) Slides were subjected to repeated cycles of staining and imaging with fluorescent primary antibodies and DAPI. At the end of each cycle, fluorescent signal was removed by a chemical bleaching process, and slides were again imaged, before proceeding to the next round of this iterative procedure. After the final antibody stain (#15 Sma), slides were analyzed with a series of histochemical stains. (b) A set of tiling images spanning each tissue section was initially generated by the microscope system. The tiling images were then computationally ‘stitched’ together to produce a single image per staining cycle for each sample. (c) Image registration was performed to align images from the same tissue section across cycles. Mean intensities of the DAPI signal from all immuno-fluorescence images are shown from before (Unregistered) and after (Registered) the image registration procedure was completed. (d) Following registration, signal intensities from the relevant channels for each image (columns) in the MMMP series were extracted for each pixel (rows) within the tissue section and compiled into a large data matrix of in situ molecular profiles. Image collected and cropped by CiteAb from the following open publication (https://dx.plos.org/10.1371/journal.pone.0128975), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Dkk-3 by Immunohistochemistry Loss of DKK3 protein expression in human breast cancer.(A) Normal mammary epithelial cells showing moderate, predominantly cytoplasmic, DKK3 immunoreactivity whereas (B) primary antibody negative control is free of signal. (C-E) Weak DKK3 protein expression is observed in breast tumor samples, with lowest intensity in IHC-defined (C) TNBC cases compared to (D) HER2-positive and (E) luminal carcinomas (representative images). (F) Box plot analysis demonstrating a significant down-regulation of DKK3 in tumors of the TNBC (n = 54), the HER2-positive (n = 47) and luminal subtype (n = 362) compared to normal breast tissues (n = 11). Horizontal lines: grouped medians. Boxes: 25-75% quartiles. Vertical lines: range, minimum and maximum. * P < 0.05, *** P < 0.001, IRS: immunoreactive score. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/27467270), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Dkk-3 by Western Blot Stable DKK3 re-expression reduces cell growth of basal-like but not luminal-like breast cancer cell lines.(A) Re-expression of DKK3 protein as well as secretion of DKK3 was detected by western blot. Western blot analysis was performed on total cell lysates and corresponding cell culture supernatants of three stably transfected MDA-MB-436, MDA-MB-231, MDA-MB-453 and MCF-7 DKK3 and mock clones respectively. beta -actin served as a loading control. (B) All western blots depicted in A were evaluated densitometrically. In concordance, mock clones were negative for DKK3 protein whereas expression was elevated in total cell lysates of DKK3 clones. Moreover, a strong secretion of DKK3 into the cell culture supernatant could only be detected in DKK3 clones. The identical clones were used for the following functional assays. (C-D) Re-expression of DKK3 significantly reduced cell growth in basal-like (C, MDA-MB-436 and MDA-MB-231) but not luminal-like breast cancer cell lines (D, MDA-MB-453 and MCF-7). Box plots demonstrate the median cell number after 96 h cell growth of triplicate experiments. Cell growth suppression was possibly mediated by a DKK3-induced apoptosis, which was much more pronounced in breast cancer cell lines of the basal (E) than of the luminal subtype (F). Box plots demonstrate the median apoptosis rate of triplicate experiments. Horizontal lines: grouped medians. Boxes: 25-75% quartiles. Vertical lines: range, minimum and maximum. ns: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/27467270), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Dkk-3 by Immunohistochemistry Loss of DKK3 protein expression in human breast cancer.(A) Normal mammary epithelial cells showing moderate, predominantly cytoplasmic, DKK3 immunoreactivity whereas (B) primary antibody negative control is free of signal. (C-E) Weak DKK3 protein expression is observed in breast tumor samples, with lowest intensity in IHC-defined (C) TNBC cases compared to (D) HER2-positive and (E) luminal carcinomas (representative images). (F) Box plot analysis demonstrating a significant down-regulation of DKK3 in tumors of the TNBC (n = 54), the HER2-positive (n = 47) and luminal subtype (n = 362) compared to normal breast tissues (n = 11). Horizontal lines: grouped medians. Boxes: 25-75% quartiles. Vertical lines: range, minimum and maximum. * P < 0.05, *** P < 0.001, IRS: immunoreactive score. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/27467270), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Dkk-3 by Western Blot Ectopic expression of DKK3 in MB231 cells disrupted Wnt signalling. (A) Subcellular localization of beta -catenin and active-beta -catenin by immunofluorescence staining. (B) Western blot analysis of beta -catenin, its downstream targets, JNK and EMT markers. (C) RT-PCR analysis of representative stem cell markers in DKK3-transfected MB231 cells. *Indicates significantly down-regulated bands. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/23890219), licensed under a CC-BY license. Not internally tested by R&D Systems.