Nectin-1 was detected on the surface of cells by staining them with anti-nectin-1 antibody CK41 conjugated to PE (BD Biosciences). HVEM was detected on the surface of cells using the anti-HVEM polyclonal antibody, R140, a gift from Gary Cohen (University of Pennsylvania), and a fluorescein isothiocyanate (FITC)-conjugated anti-rabbit secondary antibody (Thermo Fisher). Briefly, 1 × 106 cells were plated into 10-cm dishes. The next day, cells were lifted from the dishes with 1× PBS containing 5 mM EDTA. Cells were then pelleted, resuspended in 300 μl FACS buffer (1× PBS, 2% FBS, 1 mM EDTA), and divided evenly between microcentrifuge tubes for mock, PE-isotype, or PE-anti-nectin-1 treatment. Cells were incubated with 1 μg of antibody for 30 min on ice with agitation every 10 min. Similarly, cells were divided for mock, isotype (anti-gB R68 polyclonal antibody), or R140 anti-HVEM treatment. Cells were incubated with 5 μg of antibody. Mock-, isotype-, and R140-labeled cells were then incubated with a FITC-conjugated anti-rabbit secondary antibody (Thermo Fisher). After 30 min, cells were pelleted and washed three times with FACS buffer, resuspended, and then immediately analyzed by flow cytometry (FACSCalibur).
Fitc conjugated anti rabbit secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The FITC-conjugated anti-rabbit secondary antibody is a laboratory reagent used to detect and visualize primary antibodies raised in rabbits. It is conjugated with the fluorescent dye fluorescein isothiocyanate (FITC), which emits green fluorescence upon excitation, allowing for the detection and localization of target proteins in various applications, such as immunofluorescence microscopy, flow cytometry, and Western blotting.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Cy3 conjugated anti mouse secondary antibody, by Jackson ImmunoResearch (2 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Fixation permeabilization solution, by Thermo Fisher Scientific (1 mentions)
Confocal microscope, by Zeiss (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Rabbit anti cleaved caspase 3 polyclonal antibody, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Santa Cruz Biotechnology (1 mentions)
Tunel kit, by Roche (1 mentions)
Anti at1, by Santa Cruz Biotechnology (1 mentions)
Anti α mhc, by Abcam (1 mentions)
Klf4 antibody, by Santa Cruz Biotechnology (1 mentions)
Thy1.1 monoclonal antibody, by Cell Signaling Technology (1 mentions)
C ebpδ, by Santa Cruz Biotechnology (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (1 mentions)
10 protocols using fitc conjugated anti rabbit secondary antibody
1
Detecting Nectin-1 and HVEM Expression
2
Flow Cytometric Analysis of PGC-1β in Monocytes
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Peripheral blood CD14+ monocytes were stained first with PE‐conjugated anti‐CD14 monoclonal antibody (BD Biosciences) for cell surface antigen. Cells were washed twice with staining wash buffer and centrifuged (1,000 revolutions per minute for 5 minutes) to pellet the cells. They were then resuspended with 100 μl of fixation/permeabilization solution (eBioscience) for 30 minutes at 4°C to expose intracellular antigen. Cells were washed twice with 500 μl of wash buffer and suspended with 100 μl of permeabilization buffer mixed with 1 μl of rabbit anti‐human/mouse PGC‐1β antibody (Bioss) in the dark for 30 minutes at room temperature. Next, they were washed twice and resuspended with 100 μl of permeabilization buffer mixed with 1 μl of FITC‐conjugated anti‐rabbit secondary antibody (Invitrogen) in the dark for 20 minutes at room temperature. Stained cells were washed with permeabilization buffer and resuspended with 200 μl of phosphate buffered albumin (0.5% BSA and 0.05% NaN3 in PBS) before flow cytometric analysis. In each case, staining was compared to that of the appropriately labeled isotype control antibody.
3
Quantifying Phosphorylated LRP6 in Cells
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Cells were fixed with 4% paraformaldehyde. After washing and blocking with PBS containing 2% bovine serum albumin (Sigma-Aldrich), cells were incubated with anti-phospho-LRP6 (Ser 1490) (1:100, Bioss Inc., Woburn, MA, USA) in 4°C overnight. They were washed in PBS and then incubated with FITC-conjugated anti-rabbit secondary antibody (1:1000; Invitrogen, Carlsbad, CA, USA). After washing in PBS, the cells were incubated with 4′,6-diamidino-2-phenylindole (10 μl/ml; Sigma-Aldrich) and observed under a confocal microscope (Zeiss, Munich, Germany).
4
Immunofluorescence Analysis of Cultured HPMCs
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Cultured HPMCs were fixed in 4% paraformaldehyde and permeabilized with 0.25% Triton X-100. After blocking in 2% BSA (Invitrogen) for 30 min, the cells were incubated with primary antibodies at 4 °C overnight and then incubated with FITC-conjugated anti-rabbit secondary antibody (1:1000, Invitrogen) and Cy3-conjugated anti-mouse secondary antibody (1:1000, Jackson ImmunoResearch). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, 1:5000, Sigma) for 5 min. Positive staining was observed under a fluorescence microscope (Olympus, Japan).
5
Apoptosis Detection in Spinal Cord
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A TUNEL (terminal deoxynucleotidyl-transferasemediated dUTP nick-end labeling) assay is the most commonly used technique for examining apoptosis via DNA fragmentation. In situ detection of apoptosis in spinal cords was performed through staining using a TUNEL kit from Roche (Mannheim, Germany) to modify genomic DNA utilizing terminal deoxynucleotidyl transferase (TdT), according to the manufacturer's instructions.
For immunofluorescence studies, the sections were washed three times in PBS and incubated in anti-Cleaved Caspase 3 rabbit polyclonal antibody (Cell Signaling Technology, USA) for 3 h at room temperature, and then incubated in FITC-conjugated anti-rabbit secondary antibody (Invitrogen, USA) for 1 h at room temperature. Then, cell nuclei were labeled with DAPI (Santa Cruz Biotechnology, USA), and examined under a laser scanning spectral confocal microscope by two independent observers.
Average percentage of TUNEL-positive or Cleaved Caspase 3-positive motor neurons in the anterior spinal cord of the three sections were counted for comparisons among the groups. Positively stained fluorescein-labeled cells were visualized and photographed by fluorescence microscopy.
For immunofluorescence studies, the sections were washed three times in PBS and incubated in anti-Cleaved Caspase 3 rabbit polyclonal antibody (Cell Signaling Technology, USA) for 3 h at room temperature, and then incubated in FITC-conjugated anti-rabbit secondary antibody (Invitrogen, USA) for 1 h at room temperature. Then, cell nuclei were labeled with DAPI (Santa Cruz Biotechnology, USA), and examined under a laser scanning spectral confocal microscope by two independent observers.
Average percentage of TUNEL-positive or Cleaved Caspase 3-positive motor neurons in the anterior spinal cord of the three sections were counted for comparisons among the groups. Positively stained fluorescein-labeled cells were visualized and photographed by fluorescence microscopy.
6
Cytometry Analysis of NOX4 and DUOX2
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Unpermeabilized cells were stained for NOX4 (clone 20.3) [20 (link)] or anti-HA (DUOX2) for 20min, followed by FITC-conjugated anti-rabbit secondary antibody (30min; Invitrogen), fixation, and analysis using a CytoFLEX LX cytometer.
7
Immunofluorescence Staining of COS7 and Cardiomyocytes
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COS7 cells or cardiomyocytes were fixed in 4% paraformaldhyde at room temperature for 15 minutes, and then permeabilized with 0.25% Triton X-100 [25] . After blocking in 2% BSA (Gibco) for 30min, cells were incubated with primary antibodies: anti-AT 1 (1:100 SantaCruz), or anti-rhodopsin (1:200, Cell Signaling) at 4°C overnight and then incubated with FITC-conjugated anti-rabbit secondary antibody (1:1000, Invitrogen) and Cy3 -conjugated antimouse secondary antibody (1:1000, Jackson Immuno Research). α-MHC positive area of cardiomyocytes was analyzed by immunostaining with anti-α-MHC (1:100, Abcam). Nuclei were stained with 4, 6-diamidino-2phenylindole (DAPI, 1:5000, Sigma) for 5 min. The positive staining was observed under fluorescence microscope (Olympus, Japan).
8
KLF4 and hTERT Immunofluorescence Assay
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H322/mock, H322/c2 and H322/c5 cells were cultured in 1640 medium and plated in 6-well plates at a density of 1 × 105 cells per well; then, 2–4 glass coverslips were placed in each of the wells. The plates were incubated for 24 to 48 hours until reaching 30–40% confluence prior to fixation; they were then fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in PBS. The slides were washed three times with PBS and blocked with 5% BSA in PBS for 30 min at room temperature. The cells were incubated with the KLF4 antibody (1:100; Santa Cruz Biotechnology, sc-20691) or hTERT antibody (1:100; Santa Cruz Biotechnology; sc-68720) overnight at 4°C, which was followed by incubation with a fluorescein isothiocyanate (FITC)-conjugated anti-rabbit secondary antibody (1:100; Thermo Scientific) for 30 min in the dark. Nuclei were counterstained with DAPI for 15 min and then analyzed using a fluorescent microscope.
9
Immunofluorescent Labeling of Retinal Ganglion Cells
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Retinal ganglion cells grown on cover slips were fixed in 4% paraformaldehyde as previously described7 (link)8 9 (link). Briefly, cells were blocked in 5% BSA for 1 h at room temperature, followed by overnight incubation at 4 °C with the following primary antibodies: GPR91 polyclonal antibody (1:200, Novus Biologicals, Littleton, CO, USA), Thy1.1 monoclonal antibody (1:200), c-Fos monoclonal antibody (1:200, Cell Signaling Technology, Boston, MA, USA), C/EBP β monoclonal antibody (1:200, Novus Biologicals, Littleton, CO, USA), and C/EBP δ (1:200, Santa Cruz, CA). The cells were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit secondary antibodies, phycoerythrin PE-conjugated anti-mouse secondary antibodies or PE-conjugated anti-rabbit secondary antibodies (1:200, Invitrogen, Carlsbad, CA) for 1 h at room temperature. Fluorescence imaging and analyses were performed using a fluorescence microscope (Leica, Wetzlar, Germany).
10
Immunofluorescence Staining of Paraffin Sections
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