Porcine kidney (PK-15) cells (ATCC® CCL-33™) seeded in plates (Corning, NY, USA) were infected with the CSFV C-strain. Forty-eight hours post-infection, the cells were fixed with 80% ice-cold acetone in PBS for 30 min at −20 °C and washed with PBS twice. After blocking with 5% (w/v) nonfat milk in PBS and washing twice with PBS, the cells were incubated with different primary antibodies for 1 h at 37 °C. After rinsing with PBS twice, a 1:2000 dilution of the mAbs (3A9 and 4F7) and a FITC-conjugated goat-anti-mouse antibody (Thermo Scientific, USA) was added to each well for a 1 h incubation at 37 °C. Following three washes, the signal was visualized with a confocal laser scanning microscope (Leica, Wetzlar, Germany).
Fitc conjugated goat anti mouse antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The FITC-conjugated goat anti-mouse antibody is a secondary antibody that binds specifically to mouse primary antibodies. It is conjugated to the fluorescent dye FITC, which allows for the detection and visualization of target proteins in various immunoassay applications.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Confocal laser scanning microscope, by Leica camera (1 mentions)
Peroxidase labeled secondary antibodies, by Proteintech (1 mentions)
Myc tag antibody, by Proteintech (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Bak antibody, by Abcam (1 mentions)
Noxa antibody, by Abbkine (1 mentions)
Bromodeoxyuridine (brdu), by Thermo Fisher Scientific (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Bromodeoxyuridine (brdu), by Roche (1 mentions)
Fluorescence microscopy, by Olympus (1 mentions)
Skim milk, by Thermo Fisher Scientific (1 mentions)
Skim milk, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Lsr 2, by BD (1 mentions)
Rhodamine conjugated goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
10 protocols using fitc conjugated goat anti mouse antibody
1
Immunofluorescence Assay for CSFV Detection
2
Comprehensive Protein Expression Analysis
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Five micrograms of protein were electrophoresed in 10% SDS-PAGE gels and blotted to polyvinylidine difluoride membranes. Specific primary antibodies were detected with peroxidase-labeled secondary antibodies (ProteinTech Group Inc.) by using SuperSignal West Dura Extended Duration Substrate (Pierce Chemical) per manufacturer's instructions. The used antibodies from ProteinTech Group Inc. included myc-tag antibody (Cat. #16286-1-AP), ASS antibody (Cat. #66036-1-Ig), GAPDH antibody (Cat. #60004-1-Ig), Flag-tag antibody (Cat. #66008-3-Ig), p53 antibody (Cat. #60283-2-Ig), Bcl-2 antibody (Cat. #60178-1-Ig), PUMA antibody (Cat. #55120-1-AP), Bax antibody (Cat. #60267-1-Ig), caspase 9 antibody (Cat. #66169-1-Ig), caspase 3 antibody (Cat. #66470-2-Ig), Histone H3 antibody (Cat. #17168-1-AP), HRP-conjugated goat anti-mouse IgG (Cat. #SA00001-1) and HRP-conjugated goat anti-rabbit IgG (Cat. #SA00001-2). FTL antibody (Cat. #ab201975) was from Abcam Inc.. p53AIP1 antibody (Cat. #ABP56144) was from Abbkine Inc., Noxa antibody (Cat. #ab13654) was from Abcam Inc.. Bak antibody (Cat. #ab69404) was from Abcam Inc.. TRITC conjugated goat anti-rabbit antibody (Cat. #A16101) was from ThermoFisher Scientific Inc.. FITC conjugated goat anti-mouse antibody (Cat. #62-6511) was from ThermoFisher Scientific Inc..
3
BrdU Incorporation Assay for Cell Proliferation
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LacZ control shRNA- or DARPP-32 shRNA-transduced DMS-53 cells were plated at a density of 1 × 105 cells per 60-mm plate. After overnight incubation, 30 µM BrdU (Sigma-Aldrich) containing fresh medium was added to the plate for 30 min. BrdU-labelled cells were then harvested, fixed, and incubated with primary monoclonal mouse antibodies (Roche) that recognise bound BrdU in the DNA. BrdU-positive cells were detected using flow cytometry following incubation with secondary FITC-conjugated goat anti-mouse antibody (Invitrogen, Carlsbad, CA). Subsequently, propidium iodide staining was performed to differentiate between viable and dead cells. FlowJo software was used for analysis. The average of three separate experiments has been documented in the graphs while one representative experiment has been depicted in the main figure.
4
Immunofluorescence analysis of parasites
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Purified parasites were fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS for 30 min at room temperature [17 (link)]. Slides were incubated with anti-rPbTIP antisera (1:100) for 1 hr at 37°C. The slides were then washed twice with PBS and incubated with FITC-conjugated goat anti-mouse antibody (1:500) (Invitrogen) for 30 min at 37°C. Nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI; Invitrogen) 1 μg/ml. Stained parasites were mounted with ProLong® Gold antifade reagent (Invitrogen), and captured on fluorescence microscopy (Olympus, Tokyo, Japan).
5
Immunofluorescence Microscopy of Plasmodium Stages
6
Flow Cytometric Analysis of HIV Receptors
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Cells were aliquoted equally into 13 mm tubes and washed in PBS with 2% FBS. Pelleted cells were then resuspend and stained with particular antibodies on ice for 30 min. CCR5 staining was done with conjugated anti-CCR5 monoclonal antibody 2D7-FITC [fluorescein isothiocyanate] (BD Pharmingen). The anti-CD4 staining was done with mAb #19 [88 (link)], and CXCR4 staining was done with mAb 12G5 [88 (link)] followed by secondary staining with a FITC-conjugated goat anti-mouse antibody (1:40 dilution; Invitrogen). For the in vitro and in vivo primary CD4 T cell staining the anti-CD4 was labeled with BV421, clone OKT4, (BioLegend, San Diego, CA) and the anti-p24 Gag antibody, clone KC57, was labeled with RD1 (Beckman Coulter, Brea, CA). C34 staining was done with an anti-C34 mAb, generated at Green Mountain Antibodies (Burlington, VT) by immunizing Balb/c mice with the C34 peptide synthesized at ELIM Biopharmaceuticals (Hayward, CA). Fluorescence-activated cell sorter analysis was performed either on a Becton Dickinson FACS Calibur flow cytometer or a BD LSRII.
7
Bufalin Modulates CCRK and β-Catenin Localization
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PLC5 cells were plated on glass coverslip and cultured overnight to 30% confluence. Cells were then treated with bufalin or vehicle for 24 hours followed by fixation with 3% paraformaldehyde and permeation with 0.1% Triton X-100. Nonspecific binding sites were blocked with 1% BSA for 30 minutes. The cells were incubated with primary mouse anti-human CCRK antibody (Sigma) or rabbit anti-human β-catenin antibody (Abcam) for 1 hour, and then incubated with FITC-conjugated goat-anti-mouse antibody (Invitrogen) or rhodamine-conjugated goat-anti-rabbit antibody (Invitrogen) for 30 minutes. Nuclei were counterstained by DAPI (Invitrogen) and Images were captured using confocal microscope (Olympus).
8
Immunofluorescence Assay for Bordetella
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B. bronchiseptica harboring pIVET-IP vector or pIVET-PBB0324 was grown to mid-log phase, pelleted by centrifugation and resuspended in D-PBS(À). The resuspended cells were immobilized on a cover glass that had been previously treated with 0.1% poly-L-lysine. Unbound bacteria were removed by three washes with D-PBS(À), after which the remaining bacteria were probed with mouse monoclonal antibodies against c-Myc tag (Santa Cruz) and FITC-conjugated goat antimouse antibody (Invitrogen). Between each step, the cover glass was washed three times with D-PBS(À) containing 0.1% BSA. The bacteria were observed under a fluorescence microscope (BX51; Olympus, Tokyo, Japan).
9
Cellular Metabolic Regulation Assay
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Metformin, AICAR, doxycycline, berberine and mouse monoclonal antibody against flag were purchased from Sigma Aldrich (St. Louis, MO, USA). A769962 was from Bio‐Techne Corporation (Minneapolis, MN, USA). Mitochondria isolate kit (MitoSciences) was from Abcam (Cambridge, MA, USA). The following antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): β‐actin, AMPKα total and phospho‐T172, AMPKβ, and phospho‐ACC(S79). 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), Lipofectamine2000, mouse monoclonal antibody against mitochondrial ATP synthase β subunit, Cyanine‐3‐conjugated goat anti‐rabbit antibody and FITC‐conjugated goat antimouse antibody were from Life Technologies (Grand Island, NY, USA). Mouse monoclonal antibody against LKB1 was from Santa Cruz Technology (Santa Cruz, CA, USA). Gibson Assembly Cloning kit was purchased from New England Biolabs (Ipswich, MA, USA). mRNA and DNA extraction kits were from Qiagen (Germantown, MD, USA). Seahorse XF Glycolysis Stress Test Kit and Seahorse XF Cell Mito Stress Test Kit were purchased from Agilent (Santa Clara, CA, USA).
10
Immunofluorescence Microscopy of Cellular Organelles
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Cells were fixed in 4% paraformaldehyde and to visualise LC3, an anti-LC3 polyclonal antibody (Abgent) followed by FITC-conjugated goat anti-rabbit antibodies (Life Technologies) was used. Calreticulin polyclonal antibodies (Affinity Bioreagents) and Texas Red-conjugated goat anti-rabbit polyclonal antibodies (Jackson Immunoresearch Laboratories) were used to visualise calreticulin. FITC-labelled wheat germ agglutinin was used for cell surface staining and CD1a monoclonal antibody (BD Pharmingen) followed by FITC-conjugated goat anti-mouse antibody (Life Technologies) was used to visualise the CD1a glycoprotein. In live cells monodansylcadaverine (Molecular Probes) was used as an autofluorescent vital dye. DAPI (Sigma Aldrich) was used to visualise nuclei. At least three independent experiments were carried out according to standard and manufacturer’s recommended procedures and analysed using ApoTome Axio Observer Z1 inverted microscope (Zeiss) equipped with an AxioCam MRm Rev.3. Co-localisations were assessed with Axio Vision software, release 4.6.3 (Zeiss).
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