Anti-human KDR antibodies (BioLegend), anti-human CD34 antibodies (Beckman coulter), anti-human VE-cadherin antibodies (eBioscience), sheep anti-human CD31 antibodies (R&D systems), FITC-conjugated anti-sheep IgG antibodies (Jackson ImmunoResearch), mouse anti-human nuclei antibodies (Millipore), Cy3-conjugated anti-mouse IgG antibodies (Jackson ImmunoResearch), Alexa-Fluor 647-labelled rat anti-mouse TER-119 antibodies (BioLegend), rat anti-human CD49f antibodies (GoH3, BD), anti-human CD29 antibodies (AIIB2; developed by Caroline H. Damsky and obtained from Developmental Studies Hybridoma Bank) were used in this study. Rabbit polyclonal antibody against ACID/BASE coiled-coil peptides was produced by immunizing with ACID/BASE coiled-coil peptides as an immunogen. HRP-conjugated streptavidin was purchased from Thermo Scientific (Rockford, IL).
Cy3 conjugated anti mouse igg antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
Cy3-conjugated anti-mouse IgG antibody is a laboratory reagent used to detect and visualize mouse immunoglobulin G (IgG) in various experimental applications. The antibody is conjugated to the Cy3 fluorescent dye, which emits a red-orange fluorescent signal upon excitation, allowing for the identification and localization of mouse IgG in samples.
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Lab products found in correlation
Neurobasal a medium, by Thermo Fisher Scientific (3 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Hrp conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Draq5, by Cell Signaling Technology (1 mentions)
Anti lrp6 h 300 rabbit polyclonal sc15399, by Santa Cruz Biotechnology (1 mentions)
Cy2 conjugated anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Anti par2, by Santa Cruz Biotechnology (1 mentions)
Lsm5 confocal microscope, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
Can get signal immunostain immunoreaction enhancer solution, by Toyobo (1 mentions)
Bz 9000 fluorescence microscope, by Keyence (1 mentions)
Mab3420, by Merck Group (1 mentions)
Ab32454, by Abcam (1 mentions)
Neuron culture medium, by Fujifilm (1 mentions)
Alexa 488 conjugated anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
Acetylated tubulin monoclonal antibody, by Merck Group (1 mentions)
Ab18184, by Abcam (1 mentions)
Nis elements c er software, by Nikon (1 mentions)
8 protocols using cy3 conjugated anti mouse igg antibody
1
Multicolor Immunostaining of Endothelial Cells
2
Immunohistochemical Analysis of PAR2 Tumor Markers
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Paraffin embedded slides derived from PAR2 induced mammary gland tumors were deparaffinized and incubated in 3% H2O2. Antigen retrieval was carried out by heating (20 min) in a microwave oven in 10 mM Tris buffer containing 1 mM EDTA. After blocking the slides were incubated with the following primary antibodies: anti LRP6 (H-300 rabbit polyclonal sc15399, Santa Cruz Biotechnology, Inc. Dallas Texas, USA), anti-PAR2 (4μg/ml [SAM11] Santa Cruz), Cy2 conjugated anti-rabbit IgG, and Cy3-conjugated anti-mouse IgG antibodies (4μg/ml, Jackson Laboratories) were used as secondary antibodies. Nuclear staining was performed using DRAQ5 (4μM, Cell Signaling). Images were obtained using a Zeiss LSM 5 confocal microscope and analyzed with Zen software (Zeiss).
3
Immunofluorescence Analysis of Autophagy Proteins in HEK293 Cells
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HEK293 cells (0.5 x 106 cells) were transfected with 0.2 ug plasmids encoding HA-IKKβ, HA-IKKβ (L353A), Flag-Keap1, and GFP-LC3. Cells were grown on collagen-coated coverslips. After 18 h, cells were fixed with 4% paraformaldehyde in PBS for 30 min, and then fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 30 min at room temperature. After washing with PBS, cells were stained with anti-HA rat monoclonal antibodies or anti-Flag (M2) mouse antibodies in Can Get Signal immunostain Immunoreaction Enhancer Solution (TOYOBO) for 2 h. After washing with PBS, coverslips were incubated with Cy3-conjugated anti-mouse IgG antibodies or FITC-conjugated anti-rat IgG antibodies (Jackson ImmunoResearch Laboratories) for 2 h and were observed under BZ-9000 Fluorescence Microscope (KEYENCE).
4
Analyzing Ilf3 Mutant Neuron Development
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Dissociated AMY neurons were prepared from Ilf3+/+ and Ilf3ΔPrLD/ΔPrLD littermates at embryonic day 16-18 (E16-18). Neurons were plated at a density of 5.2 × 103 cells/cm2 onto poly-D-lysine-coated coverslips (Matsunami, Osaka, Japan) in Neurobasal-A medium (Thermo Fisher Scientific) containing B-27 supplement (Thermo Fisher Scientific), 0.5 mM glutamine, and 25% Neuron culture medium (FUJIFILM Wako Pure Chemical). Cultured neurons were incubated at 37°C in a 5% CO2 incubator. They were fixed with 3.7% formaldehyde in PBS for 10 min at 3, 5, and 7 days in vitro (DIV).
Immunostaining was performed as described above. The primary antibodies used were anti-MAP2 rabbit polyclonal antibody (ab32454, Abcam) and anti-Tau-1 mouse monoclonal antibody (MAB3420, Sigma-Aldrich). Secondary antibodies were Alexa488-conjugated anti-rabbit IgG antibody (Thermo Fisher Scientific) and Cy3-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch, West Grove, PA). Fluorescence images were acquired using the A1 confocal laser scanning microscope with a 20× or 40× objective. Serial images acquired at 0.5 μm steps were z-stacked. Concentric circles were drawn at 30 μm intervals around the nucleus for Sholl analysis, where the number of intersections was counted. One of the neurites with the strongest tau staining was determined to be the axon.
Immunostaining was performed as described above. The primary antibodies used were anti-MAP2 rabbit polyclonal antibody (ab32454, Abcam) and anti-Tau-1 mouse monoclonal antibody (MAB3420, Sigma-Aldrich). Secondary antibodies were Alexa488-conjugated anti-rabbit IgG antibody (Thermo Fisher Scientific) and Cy3-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch, West Grove, PA). Fluorescence images were acquired using the A1 confocal laser scanning microscope with a 20× or 40× objective. Serial images acquired at 0.5 μm steps were z-stacked. Concentric circles were drawn at 30 μm intervals around the nucleus for Sholl analysis, where the number of intersections was counted. One of the neurites with the strongest tau staining was determined to be the axon.
5
Zebrafish Embryo Analysis by In Situ Hybridization
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Zebrafish embryos were fixed with 4% paraformaldehyde and analyzed by whole mount in situ hybridization using the protocol described previously[44 (link)]. The following antisense riboprobes were generated in this study: spaw[45 (link)], myl7[46 (link)] and foxa3[47 (link)]. Whole mount immunofluorescence was performed to detect monocilia in Kupffer’s vesicle as described[48 (link)]. Acetylated tubulin monoclonal antibody (1:1000; Sigma) and Cy3-conjugated anti mouse IgG antibody (1:500; Jackson ImmunoResearch Laboratories) were used in this study. Images were acquired on a Zeiss LSM700 confocal microscope. Images were processed using ImageJ software (NIH).
6
Imaging Cultured Cells with Confocal Microscopy
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Images of cultured cells were recorded under a confocal microscope (A1; Nikon, Tokyo, Japan). The primary antibody for His-tagged ribosomes was anti-His tag antibody (Abcam, ab18184; 1:200), and the secondary antibody was Cy3-conjugated anti mouse-IgG antibody (Jackson ImmunoResearch, 115-165-166, 1:200).
The following is a list of instrument parts and settings used in this study. Laser lines at 405 and 561 nm were used for excitation of DAPI and Cy3, respectively. An oil-immersion objective lens (Apo TIRF 100× Oil, NA = 1.49; Nikon) was used to capture high-magnification images. The pixel size of the confocal images was set to 30 nm. The pinhole size was set to 0.3–0.6 AU. To enhance the resolution, confocal images were deconvolved by using NIS-Elements C-ER software (Nikon) in some cases. All images were recorded at RT (25.5 °C ± 0.5 °C).
The following is a list of instrument parts and settings used in this study. Laser lines at 405 and 561 nm were used for excitation of DAPI and Cy3, respectively. An oil-immersion objective lens (Apo TIRF 100× Oil, NA = 1.49; Nikon) was used to capture high-magnification images. The pixel size of the confocal images was set to 30 nm. The pinhole size was set to 0.3–0.6 AU. To enhance the resolution, confocal images were deconvolved by using NIS-Elements C-ER software (Nikon) in some cases. All images were recorded at RT (25.5 °C ± 0.5 °C).
7
Imaging Protein Interactions in HEK-293T Cells
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HEK-293T cells expressing AT1R-RLuc, AT2R-RLuc, or MasR-RLuc in the presence or the absence of ACE2-eGFP were fixed in 4% paraformaldehyde for 15 min and washed twice with phosphate-buffered saline (PBS) containing 20 mM glycine (PBS-glycine). Washed cells were then permeabilized with PBS-glycine containing 0.5% Triton X-100 for 5 min. Fixed and permeabilized cells were treated for 1 h with PBS containing 1% BSA and labeled with primary mouse anti-RLuc antibody (1/100; Millipore, MA, USA) followed by secondary Cy3-conjugated anti-mouse IgG antibody (1/200; Jackson ImmunoResearch, Philadelphia, PA, USA). Samples were washed several times and mounted with 30% Mowiol (Calbiochem/Merck Group, Darmstadt, Germany). Samples were observed using a Zeiss 880 confocal microscope. The signal from eGFP was detected by its green fluorescence and could be distinguished from the red signal observed in response to binding of Cy3-conjugated anti-mouse IgG. Colocalization was identified by yellow fluorescence. The scale bar presented in images measured 10 µm.
8
Immunofluorescence Staining for TLR5 and Claudin 2
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Frozen sections were fixed in 10% buffered formalin for 5 minutes and permeabilized with 0.5% tween-20 for 10 minutes. After rinsing sections were incubated overnight at 4°with rabbit anti-TLR5 (Invitrogen) (gift from S. Schueller), quenched with 10% donkey serum, and incubated with donkey Cy2-conjugated anti-rabbit IgG (Jackson Immunoresearch) for 60 minutes. Alternatively, sections were stained with rabbit anti-Claudin 2 antibody (Life Techologies) followed by incubation with FITC-labelled anti rabbit IgG antibody (Sigma) and counterstained with mouse anti-pan cytokeratin antibody (Sigma) followed by incubation with Cy3-conjugated anti-mouse IgG antibody (Jackson Immunoresearch). The primary antibody was not added to control sections. Sections were observed with a Zeiss LSM 710 or 510 confocal microscopes.
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