An in vivo angiogenesis assay was performed as previously described (Han et al., 2016 (link)). EPCs were treated with or without recombinant CTGF (r-CTGF; 20 ng/ml) for 24 h. Growth factor-reduced Matrigel (400 μl/plug) was thawed and mixed with EPCs (1 × 106/plug), and Matrigel supplemented only with r-CTGF in the absence of EPCs was used as a blank control. In addition, to investigate how VSMCs and stretch affect EPC differentiation, EPCs were cultured by themselves under static conditions or cocultured with VSMCs, which had cyclic stretch applied at 5% stretch magnitude and 1.25 Hz frequency. After 12 h, EPCs under different conditions were harvested by treatment with 0.25% trypsin and were mixed with Matrigel. Each plug was subcutaneously injected into the flank of 6-week-old male nude mice (n = 4 in each group). Mice were euthanized 7 days after surgery. Matrigel plugs were harvested, fixed in 10% formalin, and embedded in paraffin; multiple 5-μm-thick slices were prepared. Immunofluorescent staining was performed using an anti-CD31 antibody (Abcam) and anti-CD34 antibody (Santa Cruz).
Anti cd34 antibody
Manufactured by Santa Cruz Biotechnology
Sourced in Germany
The Anti-CD34 antibody is a laboratory reagent used in flow cytometry and immunohistochemistry applications. It is designed to detect the presence of the CD34 antigen, which is a transmembrane glycoprotein expressed on hematopoietic stem and progenitor cells. The antibody can be used to identify and characterize these cell populations in various biological samples.
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Lab products found in correlation
Anti cd31 antibody, by Abcam (1 mentions)
Fv1000 microscope, by Olympus (1 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
Vectashield antifade mounting medium with dapi, by Vector Laboratories (1 mentions)
Anti ki67 antibody, by Agilent Technologies (1 mentions)
Cb17 icr prkdcscid icricocrl, by Charles River Laboratories (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Celltrackertm cm dii, by Thermo Fisher Scientific (1 mentions)
Bx60 microscope, by Olympus (1 mentions)
Goat anti mouse igg fitc, by Boster Bio (1 mentions)
6 protocols using anti cd34 antibody
1
In Vivo Angiogenesis Assay with EPCs
2
Immunophenotyping of Adherent Cancer Stem Cells
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NACs smeared on slides and adherent CSCs cultured on cover slides that were coated with 0.1% gelatin were fixed while using 4% paraformaldehyde (Wako, Tokyo, Japan) for 20 min., washed two times with PBS, and then blocked using PBS containing 10% FBS at room temperature for one hour. The cells were then incubated overnight at 4 °C with primary antibodies, lineage cell detection cocktail-biotin antibody (Miltenyi Biotec, Bergisch Gladbach, Germany), anti-CD34 antibody (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-CD117/c-kit (Zytomed systems, Berlin, Germany). After incubation, the cells were washed three times with PBS and then incubated with secondary antibodies, Alexa Fluor 555 labeled anti rabbit IgG goat antibody (Thermo Fisher, Waltham, MA, USA), APC labeled anti-Biotin antibody (Miltenyi Biotec, Bergisch Gladbach, Germany), and PE labeled anti-mouse IgG goat antibody (BioLegend, San Diego, CA, USA) for 1 h. Thereafter, they were washed three times with PBS and then mounted with VECTASHIELD® Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). The cells were observed with the Olympus FV-1000 microscope (Olympus, Tokyo, Japan).
3
Establishing cSCC Xenografts and Evaluating AIM2 Knockdown
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Human cSCC xenografts were established as previously [36 (link)]. Six-week-old severe combined immunodeficient (SCID) mice (CB17/Icr-Prkdcscid/IcrIcoCrl) (Charles River Laboratories) were randomly allocated into AIM2 (n=8) and control siRNA group (n=8). After 72 hours of transfection of highly tumorigenic cSCC cell line (UT-SCC7) with AIM2 or control siRNA, cSCC cells (5×106) were injected subcutaneously into the back of mice. The size of tumors was measured twice a week and tumor volume was calculated with the formula V = (length × width2)/2 [38 (link)]. 21 days after the inoculation tumors were excised, embedded in paraffin and stained with hematoxylin and eosin (H&E). Proliferating cells were detected with anti-Ki-67 antibody (Dako, Glostrup, Denmark). Percentages of Ki-67-positive cells were determined by counting four distinct microscopic fields at 20× magnification (n=7-8). Effect of AIM2 knockdown on vascularization of the xenografts was assessed by IHC with anti-CD34 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) [36 (link)]. Blood vessel formation was evaluated in each sample by counting the number of CD34-positive blood vessels in four randomly selected microscopic fields at 20× magnification.
4
Quantifying Thrombosis-Induced Carotid Injury
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After completion of in vivo blood flow measurements in thrombosis mice model, the injured and contralateral non-injured carotids were excised and fixed in 10% buffered Formalin (Starplex Scientific Inc., Etobicoke, ON). These arterial segments were then embedded in paraffin, sectioned at 6 microns, and stained with hematoxylin and eosin, or an anti-CD34 antibody (Santa Cruz). Sections were visualized using an Olympus BX60 microscope (Olympus imaging America Inc., Center Valley, PA) and the computerized morphometric analyses were performed using a Retiga 2000R camera (QImaging Corporation, Surrey, BC), and Image Pro Plus 6.2 software (Media Cybernetics, Bethesda, MD).
For confocal fluorescence, 500 × 103 EPCs were labeled with an intracellular fluorescent marker (CellTrackerTM CM-DiI, Molecular Probes) according to the manufacturer’s instructions. Non-labeled PBMCs were used as negative control. Cells were then washed with PBS, resuspended in fresh media, injected intravenously, and allowed to circulate for 15 minutes before mouse carotid injury induction. The injured as well as the contralateral non-injured carotids were excised immediately after completion of blood flow measurements and immersed in liquid nitrogen. Labeled EPCs incorporated into the luminal aspect of arterial thrombi were observed on cryostat sections of 14-μm thickness using confocal microscopy [26 (link)].
For confocal fluorescence, 500 × 103 EPCs were labeled with an intracellular fluorescent marker (CellTrackerTM CM-DiI, Molecular Probes) according to the manufacturer’s instructions. Non-labeled PBMCs were used as negative control. Cells were then washed with PBS, resuspended in fresh media, injected intravenously, and allowed to circulate for 15 minutes before mouse carotid injury induction. The injured as well as the contralateral non-injured carotids were excised immediately after completion of blood flow measurements and immersed in liquid nitrogen. Labeled EPCs incorporated into the luminal aspect of arterial thrombi were observed on cryostat sections of 14-μm thickness using confocal microscopy [26 (link)].
5
Immunocytochemical Analysis of CD34 and β1 Integrin
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The isolated cells were cultured in a 6-well plate (104 cells/well) for 24 h. The cells were rinsed five times with PBS, fixed with 4% paraformaldehyde solution for 15 min at room temperature, and permeated with 0.5% Triton X-100-PBS solution for 5 min. After blocking with 3% BSA in PBS for 30 min, the cells were respectively incubated with primary antibodies to anti-CD 34 antibody (rabbit polyclonal antibody, 1:50, Santa Cruz Biotechnology, Inc.) and anti-β1 integrin antibody (rabbit polyclonal antibody, 1:50, Boster Biological Technology, Ltd.) at 4°C overnight. The primary antibody binding was detected via the corresponding goat anti-rabbit IgG: Cy3 and goat anti-mouse IgG FITC (Boster Biological Technology, Ltd.). Staining was examined under a fluorescence microscope.
6
Phagocytosis Assay of Opsonized Cells
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Macrophages derived from healthy volunteers were obtained as described previously [41] . BM HSPCs were sorted and opsonized with an anti-CD34 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Mature PB cells were opsonized as follows: neutrophils with anti-CD15 antibody, erythrocytes with anti-CD235a antibody, and platelets with anti-CD42b antibody. Macrophages were activated as described previously [41] . Opsonized HSPCs or mature blood cells were coincubated with activated macrophages in a 1:1 ratio at 37 C for 2 h, followed by mounting via cytospin preparation. After May-Gr€ unwald-Giemsa staining, the macrophages and engulfed cells were enumerated by a blinded observer. The phagocytosis index was calculated as described previously [41] .
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