Aspirin (over 99% purity) and the RANKL and TRAP Staining kits were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA, USA), Dulbecco's modified Eagle's medium (DMEM) and fluorescein isothiocyanate-conjugated secondary antibodies were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). NF-κB (anti-p50, anti-p65 and anti-IκB) and MAPKs (anti-ERK, anti-JNK and anti-p38) mouse antibodies and their phosphorylated antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
Fluorescein isothiocyanate conjugated secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fluorescein isothiocyanate-conjugated secondary antibody is a laboratory reagent used in immunoassays and other molecular biology techniques. It is a secondary antibody labeled with the fluorescent dye fluorescein isothiocyanate (FITC), which allows for the detection and visualization of target proteins or molecules.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Lsm 700, by Zeiss (2 mentions)
Aspirin, by Merck Group (1 mentions)
Rankl, by Merck Group (1 mentions)
Trap staining kit, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Mitotracker green fm, by Merck Group (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Antifade mounting medium, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Confocal laser scanning microscope, by Zeiss (1 mentions)
Anti insulin antibody, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
A confocal laser scanning microscope, by Leica Microsystems (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
12 protocols using fluorescein isothiocyanate conjugated secondary antibody
1
Osteoclastogenesis Regulation Pathway
2
Immunofluorescence Assay for Macrophages
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CPT (C5624), LPS, MitoTracker Green FM (M7514), and bafilomycin A1 were purchased from Sigma-Aldrich (St. Louis, MO, USA). The fluorescein isothiocyanate-conjugated secondary antibodies were obtained from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA) and murine recombinant proteins macrophage colony stimulating factor (M-CSF), IFN-γ, and IL-4 were purchased from Peprotech (NJ, USA).
3
Immunofluorescence Characterization of Conjunctival Cells
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Conjunctival imprints from patients with SJS and controls were fixed with 4% paraformaldehyde for 20 min at room temperature. The fixed cells were washed twice with wash buffer (0.1% bovine serum albumin (BSA) in 1X PBS), permeabilised in 1X PBS+0.1% Tween 20 and blocked with 1% BSA for 30 min at room temperature. The imprints were then incubated with primary rabbit anti vimentin/ anti-α-SMA/ mouse anti-α-tubulin (1:200), in dilution buffer (1X PBS, 1% BSA) at room temperature for 2 hours. The imprints were washed twice and incubated with corresponding fluorescein isothiocyanate-conjugated secondary antibodies (1:500, Invitrogen, Carlsbad, California, USA) for 1 hour in dark. Imprints were washed and counterstained with 4',6-diamidino-2-phenylindole for 3 min in dark. After phosphate-buffered saline Tween 20 washes, the imprints were mounted on a glass slide using antifade mounting medium (Invitrogen) and observed under confocal microscope.
4
Immunofluorescence Staining of p65
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Cells were washed twice with ice-cold PBS, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with antibodies to p65 at a dilution of 1:100 and then with fluorescein isothiocyanate-conjugated secondary antibodies (Molecular Probes) at a dilution of 1:200 as described previously [20 (link)]. Each step was performed in PBS containing 1% bovine serum albumin, and the cells were washed between steps with PBS three times for 5 min. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma). Coverslips were mounted on slides with 50% glycerine, and the cells were examined with a confocal laser-scanning microscope (LSM 700; Carl Zeiss, Oberkochen, Germany).
5
Immunofluorescence Analysis of YAP Expression
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The GC cells were digested and uniformly cultured on the coverslips at a suitable density, and treated with sitagliptin for 24 hours after the cells had fully adhered. After completing the scheduled processing, the cells in each well were fixed in cold 4% of paraformaldehyde. In order to increase the permeability of the cell membrane to the antibody, pretreated the cells with 0.3% of Triton‐X100 for another 15 minutes, and then, soaked the cells in a 5% of bovine serum albumin. Subsequently, the coverslips were completely covered with a 1:200 dilution of YAP antibody and incubated overnight in a 4°C wet box. The next day, the cells were incubated with fluorescein isothiocyanate‐conjugated secondary antibodies (Molecular Probes) for 60 minutes. Finally, 4′,6‐diamidino‐2‐phenylindole was counterstained for 5 minutes in the dark. Examined the changes in the expression of fluorescent protein positions with a fluorescence microscope (Olympus Corp.).
6
TUNEL Assay for Beta-Cell Apoptosis
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Islet-containing kidney tissues were embedded in optimum cutting temperature (OCT) compound (Tissue-Tek, CA, USA), and beta-cell apoptosis was examined in cryosections (5-µm thick) using an in situ cell death detection kit (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer’s protocol. Briefly, the tissue sections were permeabilized with 1% proteinase K for 15 min, rinsed with PBS, incubated with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reagents for 1 h at 37 °C, and washed in PBS for 5 min. Then, tissues were stained with an anti-insulin antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), a fluorescein isothiocyanate-conjugated secondary antibody, and 4′-6-diamidino-2-phenylindole (Life Technologies, Carlsbad, CA, USA). Images were captured using a laser scanning confocal microscope (Carl Zeiss).
7
Immunocytochemical Analysis of Protein Expression
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Cells were seeded on the cover-slips in 24-well plates and cultured overnight, then fixed in 4% paraformaldehyde for 20 min. After washing with PBS, cells were permeabilised in 0.25% Triton X-100 for 15 min, washed with PBS and blocked with 5% BSA for 60 min. Then cells were incubated with primary antibody overnight at 4°C. After washing with PBS, cells were incubated with fluorescein isothiocyanate conjugated secondary antibody (Life Technology, Carlsbad, CA, USA) at 37°C for 40 min. Nuclei were stained with DAPI. The protein expression was observed and analyzed using a fluorescence microscope (Nikon, Tokyo, Japan,).
8
Quantifying Eosinophil Extracellular Traps
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EETs were visualized in paraffin-embedded tissue slides (5 µm) by means of indirect immunofluorescence followed by counterstaining for DNA. After blocking and incubation with the polyclonal anti-major basic protein (MBP) (Monosan) antibody (1:50), the slides were incubated with a fluorescein isothiocyanate-conjugated secondary antibody (1:400) (Life Technologies) and the DNA was stained by incubation with propidium iodide (1 µg/ml) (Sigma Aldrich). Subsequently the slides were analyzed with a confocal laser-scanning microscope (Leica MicroSystems). Since the EETs are difficult to detect in fixed tissues, staining was performed on three different tissues for each patient.
For each patient and each piece of tissue, 5 fields were selected in the studied regions (stroma, subepithelial or at epithelial defects). In those fields, the amount of EETs was counted and normalized for the amount eosinophils. EETs were expressed as % of eosinophils generating EETs throughout the manuscript.
For each patient and each piece of tissue, 5 fields were selected in the studied regions (stroma, subepithelial or at epithelial defects). In those fields, the amount of EETs was counted and normalized for the amount eosinophils. EETs were expressed as % of eosinophils generating EETs throughout the manuscript.
9
Immunocytochemistry Staining of Podocyte Markers
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Cells were fixed with 2% paraformaldehyde plus 4% sucrose for 10 minutes at 37°C and permeabilized with 0.3% Triton X-100 (Sigma-Aldrich) for 4 minutes at room temperature. Nonspecific binding was quenched with blocking solution (2% FBS, 2% bovine serum albumin, and 0.2% bovine gelatin in PBS) for 30 minutes at room temperature. To detect ZO-1, cells were incubated (overnight at 4°C) with polyclonal rabbit anti-ZO-1 (1:200) and rinsed in PBS followed by incubation with goat antirabbit Texas Red-conjugated secondary antibody (1:200 dilution). For F-actin detection, cells were incubated with rhodamine phalloidin (20 U/mL; Molecular Probes; Thermo Fisher Scientific) after permeabilization.
Cells incubated with synaptopodin (1:100; Fitzgerald Laboratories, North Acton, MA, USA) primary antibody were incubated with fluorescein isothiocyanate-conjugated secondary antibody (Invitrogen) for 45 minutes in a dark chamber at room temperature. For the detection of nephrin, three-step immunohistochemistry was conducted. Poly-clonal guinea pig antinephrin antibody (1:100; Fitzgerald Laboratories) was incubated overnight at 4°C followed by rabbit antiguinea pig (Dako Laboratories) for 1 hour at room temperature before incubation with Alexa Fluor 688-conjugated secondary antibody.
Cells incubated with synaptopodin (1:100; Fitzgerald Laboratories, North Acton, MA, USA) primary antibody were incubated with fluorescein isothiocyanate-conjugated secondary antibody (Invitrogen) for 45 minutes in a dark chamber at room temperature. For the detection of nephrin, three-step immunohistochemistry was conducted. Poly-clonal guinea pig antinephrin antibody (1:100; Fitzgerald Laboratories) was incubated overnight at 4°C followed by rabbit antiguinea pig (Dako Laboratories) for 1 hour at room temperature before incubation with Alexa Fluor 688-conjugated secondary antibody.
10
Immunolocalization of BMP2 in Embryos
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The embryos were washed three times with phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA, Sigma-Aldrich) and fixed in 4% paraformaldehyde for 30 min. Fixed embryos were washed three times in PBS containing 0.5% BSA, used immediately, or stored at 4°C in embryo storage buffer (PBS + 0.9% sodium azide) for up to 1 week. Fixed embryos were permeabilized with 0.01% Triton X-100 for 30 min and washed three times with PBS containing 0.5% BSA before being blocked in a 5% BSA/PBS solution for 1 h. Embryos were incubated with a primary antibody against BMP2 at a 1:500 dilution in 5% BSA/PBS overnight at 4°C. Finally, these embryos were washed three times for 20 min in 0.5% BSA/PBS containing 0.05% Tween 20 (0.5% BSA/PBST) and incubated with a fluorescein isothiocyanate-conjugated secondary antibody (1:200, Invitrogen, Carlsbad, CA, United States) for 1 h. The nuclei were stained with 1 mg/mL 40′, 6′-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) for 10 min. Embryos at the 8-cell stage were chosen as negative controls. Images were taken on an Olympus FV1000 confocal microscope and processed using Adobe Photoshop. The same experiment was independently repeated three times, each time in triplicate, and 20–30 embryos of different developmental stages were examined each time.
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