The live expression of Collagen I (COL1A1), Collagen III (COL3A1) and Tenascin-C (TNC) were visualized as previously described [50 (link)]. Briefly, four treatment groups (as described previously; control, FGF2, GDF5 and FGF2 + GDF5) of MSCs were seeded and cultured on the PCL scaffolds for 14 days. The scaffolds were washed with PBS (5 min.), fixed with 4% paraformaldehyde (Wako) for 20 minutes, and then blocked with 0.3% Triton-X (Sigma-Aldrich, St. Louis, MO, USA). After PBS washing, the scaffolds were incubated in 1% BSA (10 minutes; Fisher), then 10% normal goat serum (45 minutes at room temp.; Jackson ImmunoResearch), with PBS washing between each step. Primary antibodies (mouse; Collagen I (1:250, Sigma), Tenascin-C (1:250, Abcam), Collagen III (1:200, Abcam)) were then added to the respective wells and incubated overnight at 4°C. The secondary antibody (goat anti-mouse Cy3, Jackson ImmunoResearch) was added to each scaffold at a concentration of 1:500. Each scaffold was counterstained for 10 minutes at room temperature using DAPI (Sigma-Aldrich, St. Louis, MO, USA) at a 1:500 concentration. Images of each scaffold were then obtained using confocal microscopy (Zeiss LSM 780, Germany). Red pixels represent expression levels of each factor of interest, while blue represents the DAPI nuclear stain. Cy3 expression was quantified using Image J software (NIH).
Goat anti mouse cy3
Manufactured by Jackson ImmunoResearch
Sourced in United States, United Kingdom, Germany
Goat anti-mouse Cy3 is a secondary antibody reagent used in immunoassays and other applications that require the detection of mouse primary antibodies. The Cy3 fluorescent dye is conjugated to the goat anti-mouse antibody, allowing for visualization and detection of the target antigen.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit cy3, by Jackson ImmunoResearch (8 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Goat anti mouse fitc, by Jackson ImmunoResearch (3 mentions)
Goat anti rabbit fitc, by Jackson ImmunoResearch (3 mentions)
Goat anti rabbit alexa 488, by Jackson ImmunoResearch (3 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit alexa fluor 647, by Jackson ImmunoResearch (2 mentions)
Rat anti gfp, by Nacalai Tesque (2 mentions)
Mouse anti γ tubulin, by Merck Group (2 mentions)
Mouse anti gfp and rabbit anti gfp, by Thermo Fisher Scientific (2 mentions)
Rabbit anti γh2avd, by Rockland Immunochemicals (2 mentions)
Rabbit anti phospho histone h3 ph3, by Merck Group (2 mentions)
Goat anti rat fitc, by Jackson ImmunoResearch (2 mentions)
Goat anti rabbit alexa 488, by Thermo Fisher Scientific (2 mentions)
Hoechst 33258, by Thermo Fisher Scientific (2 mentions)
Rat anti 5 ht, by Abcam (2 mentions)
Rabbit anti vinculin, by Thermo Fisher Scientific (2 mentions)
51 protocols using goat anti mouse cy3
1
Visualizing Extracellular Matrix Proteins in MSCs
2
Binding of Chimeric Receptors to HCMV Particles
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Example 8
Binding of Chimeric Receptors to HCMV Particles:
To assess the binding of Fc-Proteins to virus particles, HFFs were seeded at a density of 40,000 cells per well on IBIDI plates 1 day prior to infection. Virus preparations were pre-incubated with Fc-fusion proteins at a final concentration of 500 ng/ml for 2 h at 37° C. The virus/Fc-protein mixtures were incubated with the cells for 1.5 h on ice. Before fixation with 80% acetone, the cells were washed once with MEM. For staining of viral particles mouse hybridoma recognizing the abundant viral protein pp150 (generously provided by W. Britt, Sanchez 2000) used. As a secondary antibody goat anti-mouse Cy3 (Jackson Immuno Research was used. Visualization of bound Fc-proteins was achieved by applying anti-human Alexa488 (Invitrogen). For better orientation, cell nuclei were stained with DAPI. For quantification of PDGFR-alpha-Fc binding to HCMV particles, the grey values of 100 particles per condition were quantified using AxioVision Software (Zeiss).
3
Immunohistochemical Staining of TrkB and Tubulin
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For immunohistochemical stainings the following primary antibodies were used: Goat anti-TrkBECD (R&D, AF1494, 1:100); Mouse anti-tubulin βIII (R&D, TUJ-1 clone/catalog, 1:500). Fluorescent secondary antibodies were purchased from Jackson Immunoresearch: donkey anti-goat Cy3 (1:200), and goat anti-mouse Cy3 (1:200). Phalloidin-Rhodamine was used at 1 ug/ml (Sigma-Aldrich, P1951).
4
Mosquito Brain Immunostaining Protocol
5
Expressing Pore-Forming Toxin in E. coli
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Plasmid allowing the expression of PFO in E. coli was kindly offered by Daniel Portnoy (DP-4167) [42 (link)]. Plasmid GFPNMHCIIA was a gift from Robert Adelstein through Addgene (#11347) [43 (link)], plasmid tdTomato-F-Tractin [44 (link)] was kindly offered by John Hammer (NIH, Bethesda, MD, USA), and plasmid pUBC eGFP-KDEL was a gift from François-Xavier Campbell-Valois [45 (link)]. The following antibodies were used at 1/200 for immunofluorescence microscopy (IF): rabbit anti-NMHCIIA (Sigma, St Louis, MO, USA), mouse anti-NMHCIIA (Abcam, Cambridge, UK), and mouse anti-KDEL (Abcam, Cambridge, UK). Secondary antibodies were used at 1/200: goat anti-rabbit Alexa Fluor 488 (Invitrogen, Waltham, MA, USA) and goat anti-mouse Cy3 (Jackson ImmunoResearch, West Grove, PA, USA). For IF, F-actin was labeled with rhodamine phalloidin (Invitrogen, Waltham, MA, USA) and the PM with FITC-conjugated WGA (Sigma, St Louis, MO, USA) that recognizes sialic acid and N-acetylglucosaminyl sugar residues at the PM.
6
Immunohistochemical Profiling of Murine Lungs
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Mouse lungs were fixed with 1% PFA (Alfa Aesar) for 1hr at 4°C, washed and incubated in 34% sucrose solution (Sigma-Aldrich) overnight at 4°C. Lungs were embedded in Cryomatrix (Thermo Fischer) and frozen for cryostat sectioning (9 μm-thick). Sections were permeabilized using 0.5% saponin (Sigma-Aldrich), 2% BSA (Sigma-Aldrich), 1% FBS (Life Technologies) for 30 minutes at room temperature. Sections were labeled overnight at 4°C with mouse anti-human purified CD45 (HI30, Biolegend) followed by incubation for 1hr at room temperature with goat anti-mouse Cy3 (Jackson laboratory). All sections were labeled with Hoechst (Molecular Probes, Thermo Fisher) for nuclei staining 5 minutes at room temperature and mounted with Prolong diamond (Thermo scientific). Slides were imaged using a SP5 (Leica) and analyzed with Fiji software.
7
Phagocytosis and Cell-Cell Interaction Assays
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Cells were grown in 1 cm2 wells with 500 µl culture medium for 24 h, washed once with medium and then incubated with 50 µl FITC-latex beads (0.1 µm particle size, Cayman Chemical Company, Ann Arbor USA) for 12 h. Alternatively, cells were incubated for 6 h with isolated PBLs (small cell suspension fraction of previously cryo-preserved peripheral blood mononuclear cells (PBMC) isolated from buffy-coats, according to standard Ficoll density gradient protocols). After incubation, cells were washed two times in PBS, detached with trypsin/EDTA, centrifuged at 600 rpm for 3 min and deposited onto glass slides using a cytocentrifuge (Shandon, Pittsburgh, PA, USA). Then the slides were fixed in 100% ethanol for 1 min and air-dried. PBL incubated cells were counterstained with Hematoxylin. Latex bead incubated cells were stained with anti-HLA-DR and the secondary antibody goat anti mouse-Cy3 (Jackson Immunoresearch, Newmarket, UK) according to standard immunocytochemical protocols, counterstained with DAPI (1 µg/ml) and mounted with Vectashield (Vector Laboratories, Burlingame, USA). The slides were analyzed using an Axioscope-2 microscope (Zeiss, Germany) equipped with appropriate filter sets (AHF, Tübingen, Germany), the CCD camera Jai-M 300 and the ISIS software from Metasystems (Altlussheim, Germany).
8
Ovarian Germline Cell Staining Protocol
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Ovaries were dissected in cold phosphate-buffered saline (PBS), fixed with 4% formaldehyde/PBS at room temperature and stained with anti-Flag antibodies (Sigma-Aldrich, MAB3118) overnight at 4°C. Anti-FLAG staining was visualized using goat anti-mouse Cy3 (Jackson ImmunoResearch 715-165-151) for 2 h at room temperature and DNA was counterstained using either Propidium Iodide or DAPI. For actin staining, Phalloidin 488 (Invitrogen A12379) was used. 3D confocal stacks were captured on a Leica confocal SP4 and are presented as maximum intensity Z-projections. Nuclear diameters of the stage 8 germline cells were measured using the Fiji image processing program (Schindelin et al., 2012 (link)). Data were analyzed using Microsoft Excel and GraphPad Prism 6.
9
Immunofluorescence Antibody Staining Protocol
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The following primary antibodies diluted in PBST were used in these experiments: mouse anti-Delta, mouse anti-Arm (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), 1:200; mouse anti-GFP and rabbit anti-GFP (Molecular Probes, Eugene, OR, USA), 1:1000; rat anti-GFP (Nacalai Tesque Inc., Kyoto. Japan), 1:1000; rabbit anti-γH2AvD (Rockland, Gilbertsville, PA, USA) 1:2000; rabbit anti-pS/TQ (Cell Signaling Technologies, Danvers, MA, USA), 1:1000; rabbit anti-phospho-histone H3 (PH3, Millipore, Billerica, MA, USA), 1:1000; mouse anti-γ-tubulin (Sigma-Aldrich), 1:1000; rabbit anti-β-gal (Upstate Biotechnology Inc., Lake Placid, NY, USA), 1:1000; and anti-CCleaved caspase-3 (Cell Signaling Technologies), 1:1000; rabbit anti-pJNK antibody (Cell Signaling Technologies). The following secondary antibodies diluted in PBST were used: goat anti-rabbit FITC (Jackson ImmunoResearch, West Grove, PA, USA), 1:400; goat anti-rabbit Cy3 (Jackson ImmunoResearch), 1:400; goat anti-mouse FITC (Jackson ImmunoResearch), 1:400; goat anti-mouse Cy3 (Jackson ImmunoResearch), 1:400; goat anti-rat FITC (Jackson ImmunoResearch), 1:400, goat anti-rabbit Alexa Fluor® 647 (Jackson ImmunoResearch), 4′,6-diamidino-2-phenylindole (DAPI, Molecular Probes), 1:1000.
10
Quantifying CDV-Infected Cell Rates
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For the detection of infected cells and quantification of the infectivity rate, respectively, cells were labeled using a monoclonal mouse anti-CDV-specific antibody (clone D110; kindly provided by Prof. A. Zurbriggen, University of Bern, Switzerland). Briefly, cells were transferred to a glass slide by cytospin centrifugation (250×g, 5 minutes). After fixation with paraformaldehyde (4%) for 30 minutes at room temperature, cells were washed with phosphate buffered saline Triton X (PBST). Non-specific blocking was performed with goat and horse serum (5% each) for 20 minutes. Subsequently, cells were incubated with the primary antibody (dilution 1∶100) for 4 hours at room temperature, followed by incubation with the secondary antibody (goat anti-mouse Cy3; 1∶100; Jackson, ImmunoResearch, Dianova, Germany) for 1 hour at room temperature in a dark chamber. For counterstaining cells were incubated with bisbenzimidine (1∶100; Sigma-Aldrich, Germany) for 15 minutes at room temperature. The percentages of CDV-infected cells were determined at 24, 72 and 120 hpi in duplicates by immunofluorescence microscopy (Olympus IX-70, Olympus Life Science Europe GmbH, Germany).
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