Journal: eLife

Article Title: Inflammasome activation leads to cDC1-independent cross-priming of CD8 T cells by epithelial cell-derived antigen

doi: 10.7554/eLife.72082

Figure Lengend Snippet: ( A ) Overview of experimental setup for analyzing OT-I responses to OvaFla production in IECs. ( B ) Quantification of OT-Is as a percent of total CD8 + T cells per spleen (left) and mesenteric lymph node (right). ( C ) Total number of OT-Is per spleen (left) and mesenteric lymph node (right). ( D ) Total number of CD62L – CD44 + OT-Is per spleen (left) and mesenteric lymph node (right). Samples with fewer than 20 OT-Is were excluded from CD62L, CD44 calculations. Tissues were harvested and analyzed at day 5 post tamoxifen chow start. Data are pooled from three biological replicates, and each dot represents an individual mouse. Data shown as mean ± SD. Significance calculated using one-way ANOVA and Tukey’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001). Only p values between wild-type (WT) and other experimental groups are shown. See for exact p values. Figure 3—source data 1. Statistical data for .

Article Snippet: Antibody , Anti-mouse CD44 BB515 (rat monoclonal) , BD , Clone: IM9; Cat#: 564587 , FC(1:300).

Techniques:

Journal: eLife

Article Title: Inflammasome activation leads to cDC1-independent cross-priming of CD8 T cells by epithelial cell-derived antigen

doi: 10.7554/eLife.72082

Figure Lengend Snippet: ( A ) Schematic depicting the production and analysis workflow of chimeric bm1 + OvaFla mice (left). At the right, an illustration of either wild-type (WT) OvaFla mice (left of the dashed line) or Nlrc4 –/– OvaFla mice (right of the dashed line) following lethal irradiation and reconstitution with bone marrow from B6.SJL mice. ( B ) Quantification of OT-Is as a percent of total CD8 + T cells (left), the total number of OT-Is (middle), and the total number of CD62L – CD44 + OT-Is (right) in the spleen. ( C ) Quantification of OT-Is as a percent of total CD8 + T cells (left), the total number of OT-Is (middle), and the total number of CD62L – CD44 + OT-Is (right) in the mesenteric lymph nodes. Tissues were harvested and analyzed at day 5 post tamoxifen chow start. (B–C) Data are pooled from three biological replicates, and each dot represents an individual mouse. Data shown as mean ± SD. Significance calculated using one-way ANOVA and Tukey’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001). Only p values between WT and other experimental groups are shown. See for exact p values. Figure 4—source data 1. Statistical data for .

Article Snippet: Antibody , Anti-mouse CD44 BB515 (rat monoclonal) , BD , Clone: IM9; Cat#: 564587 , FC(1:300).

Techniques: Irradiation

Journal: eLife

Article Title: Inflammasome activation leads to cDC1-independent cross-priming of CD8 T cells by epithelial cell-derived antigen

doi: 10.7554/eLife.72082

Figure Lengend Snippet: ( A ) Percent of CD45 + cells that are conventional type one dendritic cells (cDC1s) in bm1 chimera mice that received either Batf3 + or Batf3 – donor bone marrow. ( B ) Quantification of OT-Is as a percent of total CD8 + T cells (top), the total number of OT-Is (middle), and the total number of CD62L – CD44 + OT-Is (bottom) in the spleen. ( C ) Quantification of OT-Is as a percent of total CD8 + T cells (top), the total number of OT-Is (middle), and the total number of CD62L – CD44 + OT-Is (bottom) in the mesenteric lymph nodes. ( D ) Quantification of OT-Is that have out-diluted the CellTrace Violet dye in the spleen (top) and mesenteric lymph nodes (bottom). ( E ) Percent of CD62L – CD44 + OT-Is in the mesenteric lymph node that are CCR9 + . Tissues were harvested and analyzed at day 5 post tamoxifen chow start. Samples with fewer than 20 OT-Is were excluded from CD62L, CD44, and CCR9 calculations. (A) Data are from a single experiment. (B–D) Data are pooled from three biological replicates. (E) Data are pooled from two biological replicates. Each dot represents an individual mouse. Data shown as mean ± SD. Significance calculated using one-way ANOVA and Šídák’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001). See for exact p values. Figure 5—source data 1. Statistical data for .

Article Snippet: Antibody , Anti-mouse CD44 BB515 (rat monoclonal) , BD , Clone: IM9; Cat#: 564587 , FC(1:300).

Techniques:

Journal: eLife

Article Title: Inflammasome activation leads to cDC1-independent cross-priming of CD8 T cells by epithelial cell-derived antigen

doi: 10.7554/eLife.72082

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-mouse CD44 BB515 (rat monoclonal) , BD , Clone: IM9; Cat#: 564587 , FC(1:300).

Techniques: Generated, CRISPR, Purification, Enzyme-linked Immunosorbent Assay, Software

Journal: Chinese Medical Journal

Article Title: Role of Triggering Receptor Expressed on Myeloid Cell-1 Expression in Mammalian Target of Rapamycin Modulation of CD8 + T-cell Differentiation during the Immune Response to Invasive Pulmonary Aspergillosis

doi: 10.4103/0366-6999.205850

Figure Lengend Snippet: IL-12 increases the expression of mammalian target of rapamycin and S6 kinase, which was blocked by rapamycin. The peripheral blood mononuclear cells obtained from IPA mice, CTX + IPA mice, CTX + IPA mice treated with IL-12 or rapamycin, and control animals were detected using flow cytometry 7 days after intranasal inoculation of Aspergillus fumigatus . Side scatter and CD8a were used to gate on CD8 + T-lymphocytes, and CD44 + CD45 + CD62 +/− cells represent CD8 + effector memory T-cells. The data are presented as the mean ± standard deviation. * P < 0.05. CTX: Cyclophosphamide; IPA: Invasive pulmonary aspergillosis; IL: Interleukin.

Article Snippet: Cells were then labeled with the following fluorescence-conjugated monoclonal antibodies: anti-rat CD45 PE (12-0451-81, eBioscience, San Diego, CA, USA), anti-rat CD8a APC (17-0081-81, eBioscience), anti-rat CD44 PE (25-0441-81, eBioscience), and anti-rat CD62L (104432, Biolegend).

Techniques: Expressing, Control, Flow Cytometry, Standard Deviation

Journal: Chinese Medical Journal

Article Title: Role of Triggering Receptor Expressed on Myeloid Cell-1 Expression in Mammalian Target of Rapamycin Modulation of CD8 + T-cell Differentiation during the Immune Response to Invasive Pulmonary Aspergillosis

doi: 10.4103/0366-6999.205850

Figure Lengend Snippet: The proportion of CD8 + effector memory T-cells, expression of interferon-γ, and sTREM-1 levels were significantly increased by IL-12 stimulation but significantly decreased by rapamycin treatment. The peripheral blood mononuclear cells obtained from IPA mice, CTX + IPA mice, CTX + IPA mice treated with IL-12 or rapamycin, and control animals were detected using flow cytometry 7 days after Aspergillus fumigatus intranasal inoculation. Side scatter and CD8a were used to gate on CD8 + T-lymphocytes, and CD44 + CD45 + CD62 +/- cells represented CD8 + effector memory T-cells. The data are presented as the mean ± standard deviation. * P < 0.05. CTX: Cyclophosphamide; IPA: Invasive pulmonary aspergillosis; IL: Interleukin; sTREM-1: Soluble triggering receptors expressed on myeloid cell-1.

Article Snippet: Cells were then labeled with the following fluorescence-conjugated monoclonal antibodies: anti-rat CD45 PE (12-0451-81, eBioscience, San Diego, CA, USA), anti-rat CD8a APC (17-0081-81, eBioscience), anti-rat CD44 PE (25-0441-81, eBioscience), and anti-rat CD62L (104432, Biolegend).

Techniques: Expressing, Control, Flow Cytometry, Standard Deviation

Journal: eLife

Article Title: Airway basal cells show regionally distinct potential to undergo metaplastic differentiation

doi: 10.7554/eLife.80083

Figure Lengend Snippet: ( A ) Diagram: strategy for labeling, isolation and scRNA-Seq analysis of BCs from Trp63 CreERT2 ; Rosa26 lox-STOP-lox-tdTomato adult tracheas. ( B ) Immunofluorescence (IF) of tracheal sections showing efficient tdTom co-labeling with TRP63 +KRT5+BCs restricted to the basal layer of the airway epithelium. ( C ) tSNE visualization of the six airway BC subpopulations identified by scRNA-Seq, colored by cluster assignment of each population annotated using established lineage-specific or proliferation markers. ( D ) tSNE visualization of airway BC scRNA-Seq, colored by expression (log2(TPM +1)) of representative marker genes of the different BC subpopulations. ( E ) Enriched KEGG gene sets in BC-1 and BC-2 subpopulations. ( F ) Left panel: Volcano plot showing differentially expressed genes in BC-1 and BC-2 (right; black dots); genes significantly enriched in BC-1 were colored in blue (-log10(p)>10; d: average expression difference >1); those significantly enriched in BC-2 were colored in orange (-log10(q)>10; d < –1). Right panel: tSNE visualization of Tgm2, Isl1, Cav1, Krt17, and Cd44 distribution in the BC subpopulations. Scale bar in ( B ): 10 μm.

Article Snippet: The following primary antibodies were used: rabbit anti-TRP63a (1:100, CST, 13,109 s); chicken anti-KRT5 (1:300, Biolegend, 905901); rabbit anti-KRT17 (1:10000, Abcam, ab53707); rabbit anti-CAV1 (1:100, Cell Signaling Technology, 3267 S); rat anti-CD44 (1:100, BD Biosciences, BD553131); rabbit anti-TGM2 (1:50, Cell Signaling Technology, 3557 S); rabbit anti-ISL1 (1:50, Abcam, ab109517); rat anti-SCGB3A2 (1:100, R&D Systems, MAB3465); goat anti-CC10 (1:1000, Santa Cruz biotechnology, sc-9772); mouse anti-ECAD (1:200, BD Biosciences, 610181); mouse anti-FOXJ1 (1:100, eBioscience, 14-9965-82); rabbit anti-Acetyl-α-TUBULIN (1:2000, Cell Signaling Technology, 5335 S); rabbit anti-VIMENTIN (1:200; Cell Signaling Technology; 5741); rat anti-KI67 (1:100, eBioscience, 14-5698-82).

Techniques: Labeling, Isolation, Immunofluorescence, Expressing, Marker

Journal: eLife

Article Title: Airway basal cells show regionally distinct potential to undergo metaplastic differentiation

doi: 10.7554/eLife.80083

Figure Lengend Snippet: Immunofluorescence of KRT5, a-SMA, BC-1 (TGM2, ISL1) and BC-2 (KRT17, CAV1, and CD44) markers in ventral and dorsal tracheal epithelium of 5dpi-Naphthalene or 7dpi-Polidocanol. Small panels are higher magnification images of the boxed areas depicting expression of each marker alone (right) or double-labeled with KRT5 (left). a-SMA identifies the smooth muscle layer in dorsal trachea (not displayed in all panels here). Scale bar: 10 μm.

Article Snippet: The following primary antibodies were used: rabbit anti-TRP63a (1:100, CST, 13,109 s); chicken anti-KRT5 (1:300, Biolegend, 905901); rabbit anti-KRT17 (1:10000, Abcam, ab53707); rabbit anti-CAV1 (1:100, Cell Signaling Technology, 3267 S); rat anti-CD44 (1:100, BD Biosciences, BD553131); rabbit anti-TGM2 (1:50, Cell Signaling Technology, 3557 S); rabbit anti-ISL1 (1:50, Abcam, ab109517); rat anti-SCGB3A2 (1:100, R&D Systems, MAB3465); goat anti-CC10 (1:1000, Santa Cruz biotechnology, sc-9772); mouse anti-ECAD (1:200, BD Biosciences, 610181); mouse anti-FOXJ1 (1:100, eBioscience, 14-9965-82); rabbit anti-Acetyl-α-TUBULIN (1:2000, Cell Signaling Technology, 5335 S); rabbit anti-VIMENTIN (1:200; Cell Signaling Technology; 5741); rat anti-KI67 (1:100, eBioscience, 14-5698-82).

Techniques: Immunofluorescence, Expressing, Marker, Labeling

Journal: eLife

Article Title: Airway basal cells show regionally distinct potential to undergo metaplastic differentiation

doi: 10.7554/eLife.80083

Figure Lengend Snippet: ( A ) IF of ISL1, CD44, CAV1 and KRT17 co-stained with KRT5 in adult human tracheal sections. Quantification of fluorescence intensity in ventral and dorsal BCs from paired regionally distinct areas. Bar graphs are mean ± SEM of the values from each BC (dots), n ≥ 227 BCs from 3 different human donors. ( B ) Differential enrichment of BC-1 and BC-2 markers in ventral and dorsal cultured human BCs, respectively. littermate control of BC-1 and BC-2 markers in 7 days submerged cultures of BCs isolated from ventral and dorsal sides of adult human donor tracheal epithelium. qPCR of ISL1, CD44, CAV1, and KRT17 expression in these cultures. Bar graphs are mean ± SEM log2(foldchange) values for each replicate culture (n=3). Data are normalized to ventral BCs. ( C ) IF of KRT13 in ALI day 28 cultures of human ventral and dorsal tracheal BCs. Scattered plot (left): percentage of KRT13 + cells per field in total ventral or dorsal BC population; mean ± SEM from 5 to 6 fields per cultures, n=3 batches. Bar graph (right): qPCR of KRT13 expression in ALI day 28 cultures from ventral and dorsal human BCs. Values (log2(foldchange)) are normalized to ventral BCs. Bars are mean ± SEM, dots representing values of each replicate. n=4 batches. Student’s t-test, **p<0.01, ****p<0.0001. Scale bars represent 10 μm.

Article Snippet: The following primary antibodies were used: rabbit anti-TRP63a (1:100, CST, 13,109 s); chicken anti-KRT5 (1:300, Biolegend, 905901); rabbit anti-KRT17 (1:10000, Abcam, ab53707); rabbit anti-CAV1 (1:100, Cell Signaling Technology, 3267 S); rat anti-CD44 (1:100, BD Biosciences, BD553131); rabbit anti-TGM2 (1:50, Cell Signaling Technology, 3557 S); rabbit anti-ISL1 (1:50, Abcam, ab109517); rat anti-SCGB3A2 (1:100, R&D Systems, MAB3465); goat anti-CC10 (1:1000, Santa Cruz biotechnology, sc-9772); mouse anti-ECAD (1:200, BD Biosciences, 610181); mouse anti-FOXJ1 (1:100, eBioscience, 14-9965-82); rabbit anti-Acetyl-α-TUBULIN (1:2000, Cell Signaling Technology, 5335 S); rabbit anti-VIMENTIN (1:200; Cell Signaling Technology; 5741); rat anti-KI67 (1:100, eBioscience, 14-5698-82).

Techniques: Staining, Fluorescence, Cell Culture, Isolation, Expressing

Journal: Cancer cell

Article Title: Liver Cancer Initiation Requires p53 Inhibition by CD44-Enhanced Growth Factor Signaling

doi: 10.1016/j.ccell.2018.05.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibody details are provided in the . table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-CD44pan (DF1485) Santa Cruz Biotechnology Cat# SC-7297; RRID: AB_627065 anti-CD44pan (IM7) ABDSerotec (Biorad) Cat# MCA4703; RRID: AB_2076194 anti-CD44v6 ABD Serotec (Biorad) Cat# MCA1967; RRID: AB_323213 anti-AFP R&D Systems Cat# AF5369; RRID: AB_2258018 anti-Ki67 Genetex Cat# GTX16667; RRID: AB_422351 anti-F4/80 (Clone: A3-1) Caltag/Thermo Fisher Cat# MA1-91124; RRID: AB_2277854 anti-Clvd.

Techniques: Recombinant, Blocking Assay, In Situ, TUNEL Assay, Polymer, Plasmid Preparation, Amplification, Staining, Extraction, Labeling, In Situ Hybridization, RNAscope, HD Assay, cDNA Synthesis, SYBR Green Assay, Negative Control, Software

Journal: bioRxiv

Article Title: PKD-1 Signaling Is Required for the Maintenance of CSCs with Epithelial-mesenchymal Plasticity in Pancreatic Neuroendocrine Tumors

doi: 10.1101/2022.02.17.480869

Figure Lengend Snippet: Identification and distribution of CD44-positive cancer stem-like cells in human pNET tissues. A . Human pNET specimens in patient 1 were co-stained with CD44 and α-SMA antibodies followed by appropriate secondary antibodies, with DAPI staining the nuclei (blue) by immunofluorescence microscopy. CD44-positive cancer cells (red) marked by yellow arrowheads were close to the α-SMA positive (green) blood vessels. B : Human pNET specimens in patient 2 were co-stained as in A . CD44-positive cancer cells (green) were indicated by yellow arrowheads, which were close to the α-SMA positive (red) blood vessels. Note: A group of CD44-positive cells appear to distribute within the α-SMA-positive vascular niche or nearby blood vessels. Shown are representative images from different individual patients. Bar = 10 or 20 µm. C . Human pNET tissues were co-stained with CD44 antibodies and CD45 antibodies followed by appropriate secondary antibodies. Overlay images were collected by immunofluorescence microscopy. CD44-positive (green, yellow arrowheads) and both CD44-positive and CD45-positive cells (orange, blue arrowheads) were observed under a fluorescence microscope. Images were acquired by a fluorescence microscope equipped with a CCD camera. Shown are representative images from two individual patients. Bar = 10 µm. Note: Cells positive with both CD44 and CD45 are smaller and regarded as lymphatic cells (orange), a subset of cancer cells are bigger and positive for CD44 but negative for CD45, indicating their stem-like phenotype.

Article Snippet: Antibodies for immunostaining included rabbit anti-human/mouse PKD-1 (SAB4502371) and mouse anti-human/mouse α-smooth muscle actin (α-SMA, A2547) (Sigma-Aldrich), mouse anti-human/rat CD44 (5640S) and HRP-linked anti-rabbit IgG (7074) (Cell Signaling Technology), CD45 monoclonal Antibody (14-9457-80), AlexaFluor 594 conjugated donkey anti-rabbit IgG (A21207), AlexaFluor 594 conjugated donkey anti-mouse IgG(A21203), AlexaFluor 594 conjugated donkey anti-goat IgG (A11058), AlexaFluor 488 conjugated donkey anti-goat IgG(A11055), AlexaFluor 488 conjugated donkey anti-rabbit IgG (A21206), and AlexaFluor 488 conjugated goat anti-mouse IgG (A11001) (Invitrogen).

Techniques: Staining, Immunofluorescence, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: PKD-1 Signaling Is Required for the Maintenance of CSCs with Epithelial-mesenchymal Plasticity in Pancreatic Neuroendocrine Tumors

doi: 10.1101/2022.02.17.480869

Figure Lengend Snippet: Stem-like phenotype of cancer cells in pNETs. A . A human pancreatic tissue control (top panel) and human pNET specimens from two individual patients (middle and lower panels) were co-stained with CD44 and α-SMA antibodies followed by appropriate secondary antibodies, with DAPI staining the nuclei (blue) by immunofluorescence microscopy. CD44-positive cancer stem-like cells were marked by yellow arrowheads, which were close to the α-SMA positive (green, red arrowheads) blood vessels. The fluorescence images were acquired by an immunofluorescence microscope equipped with a CCD camera, and representative images are shown, along with H & E staining for tumor tissue structures. Bar = 10 µm.

Article Snippet: Antibodies for immunostaining included rabbit anti-human/mouse PKD-1 (SAB4502371) and mouse anti-human/mouse α-smooth muscle actin (α-SMA, A2547) (Sigma-Aldrich), mouse anti-human/rat CD44 (5640S) and HRP-linked anti-rabbit IgG (7074) (Cell Signaling Technology), CD45 monoclonal Antibody (14-9457-80), AlexaFluor 594 conjugated donkey anti-rabbit IgG (A21207), AlexaFluor 594 conjugated donkey anti-mouse IgG(A21203), AlexaFluor 594 conjugated donkey anti-goat IgG (A11058), AlexaFluor 488 conjugated donkey anti-goat IgG(A11055), AlexaFluor 488 conjugated donkey anti-rabbit IgG (A21206), and AlexaFluor 488 conjugated goat anti-mouse IgG (A11001) (Invitrogen).

Techniques: Control, Staining, Immunofluorescence, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: PKD-1 Signaling Is Required for the Maintenance of CSCs with Epithelial-mesenchymal Plasticity in Pancreatic Neuroendocrine Tumors

doi: 10.1101/2022.02.17.480869

Figure Lengend Snippet: PKD-1 signaling in the maintenance of cancer stem-like features in pNETs. A . Distribution of PKD-1 + and CD44 + CSCs within the vascular niche. Human pNET specimens were co-stained with CD44 and PKD-1 antibodies followed by appropriate secondary antibodies, with DAPI staining the nuclei (Blue). Stem-like cells with CD44-positive (green), PKD-1-positive (red) or both positive (pink) were observed under a fluorescence microscope. A few CD44-positive cancer stem-like cells tended to accumulate near the vascular lumen (red arrow heads), and PKD-1-positive CSCs might be leaving tumor nests (stars) for the vascular lumen. The fluorescence images were acquired by an immunofluorescence microscope equipped with a CCD camera. Shown are representative images. Bar = 10 µm. B . BON and QGP-1 cells were transfected with siRNA control and siPKD-1 to knock down PKD-1. Knockdown efficiency was confirmed by Western Blots (upper panel). The control and BON cells with knocked-down PKD-1 were subjected to tumorsphere formation assays. Images were acquired by the OLYMPUS CK30 microscope. Representative images are shown for tumorsphere formation (lower panel). C . Cell lysates were extracted from BON and QGP-1 cells exposed to the vehicle control, 10 μ M LPA, 2 μ M CRT0066101, or their combinations after 24 hours. The expression levels of phosphorylated PKD-1 and PKD-1 were detected by Western blots. Shown are representative images. D . BON and QGP-1 cells were exposed to 10 μ M LPA, 2 μ M CRT0066101, or their combination for 24 hours, and total RNA was extracted for the detection of mRNA levels of genes related to stemness properties by RT-qPCR. E . Effect of PKD inhibitor in tumorsphere formation. BON cells were cultured in complete MammoCult(tm) medium with the treatment of 10 μ M LPA, 2 μ M CRT0066101, or their combination for 7 days. The mammary spheres were counted under the OLYMPUS CK30 microscope. F . Control and BON cells with PKD-1 knockdown were exposed to 10 μ M LPA, 2 μ M CRT0066101, or their combination for 24 hours, and total RNA was extracted for the detection of mRNA levels of genes related to stemness properties by RT-qPCR. Note: Triplicate experiments were performed, and the results are shown as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies for immunostaining included rabbit anti-human/mouse PKD-1 (SAB4502371) and mouse anti-human/mouse α-smooth muscle actin (α-SMA, A2547) (Sigma-Aldrich), mouse anti-human/rat CD44 (5640S) and HRP-linked anti-rabbit IgG (7074) (Cell Signaling Technology), CD45 monoclonal Antibody (14-9457-80), AlexaFluor 594 conjugated donkey anti-rabbit IgG (A21207), AlexaFluor 594 conjugated donkey anti-mouse IgG(A21203), AlexaFluor 594 conjugated donkey anti-goat IgG (A11058), AlexaFluor 488 conjugated donkey anti-goat IgG(A11055), AlexaFluor 488 conjugated donkey anti-rabbit IgG (A21206), and AlexaFluor 488 conjugated goat anti-mouse IgG (A11001) (Invitrogen).

Techniques: Staining, Fluorescence, Microscopy, Immunofluorescence, Transfection, Control, Knockdown, Western Blot, Expressing, Quantitative RT-PCR, Cell Culture

Journal: bioRxiv

Article Title: PKD-1 Signaling Is Required for the Maintenance of CSCs with Epithelial-mesenchymal Plasticity in Pancreatic Neuroendocrine Tumors

doi: 10.1101/2022.02.17.480869

Figure Lengend Snippet: Distribution of CD44-and/or PKD-1-positive cancer stem-like cells in pNET tissues. Human pNET specimens were co-stained with CD44 antibodies and PKD-1 antibodies followed by appropriate secondary antibodies, with DAPI staining the nuclei (blue). CD44-positive (green), PKD-1-positive (red) or both positive were observed under a fluorescence microscope. Cancer cells with high levels of both CD44 and PKD-1 (yellow arrowheads) likely left the tumor nests (white stars) and accumulated in the nearby vascular lumen (red arrowheads). Fluorescence images were acquired by an immunofluorescence microscope equipped with a CCD camera. Shown are representative images from two individual patients. Bar = 10 µm. Note: These are additional pictures for .

Article Snippet: Antibodies for immunostaining included rabbit anti-human/mouse PKD-1 (SAB4502371) and mouse anti-human/mouse α-smooth muscle actin (α-SMA, A2547) (Sigma-Aldrich), mouse anti-human/rat CD44 (5640S) and HRP-linked anti-rabbit IgG (7074) (Cell Signaling Technology), CD45 monoclonal Antibody (14-9457-80), AlexaFluor 594 conjugated donkey anti-rabbit IgG (A21207), AlexaFluor 594 conjugated donkey anti-mouse IgG(A21203), AlexaFluor 594 conjugated donkey anti-goat IgG (A11058), AlexaFluor 488 conjugated donkey anti-goat IgG(A11055), AlexaFluor 488 conjugated donkey anti-rabbit IgG (A21206), and AlexaFluor 488 conjugated goat anti-mouse IgG (A11001) (Invitrogen).

Techniques: Staining, Fluorescence, Microscopy, Immunofluorescence