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Fluorescent Antibody Technique

The Fluorescent Antibody Technique is a powerful analytical method that utilizes fluorescently labeled antibodies to detect and localize specific target molecules within biological samples.
This technique allows researchers to visualize the distribution and abundance of proteins, cells, or other biomolecules of interest, making it a valuable tool in a wide range of applications, such as immunohistochemistry, flow cytometry, and fluorescence microscopy.
By leveraging the specificity of antibody-antigen interactions and the sensitivity of fluorescent detection, the Fluorescent Antibody Technique enables researchers to gain valuable insights into the complex biological processes underlying health and disease.
Pubcompare.ai's AI-driven protocol comparison tool can help optimize this technique by identifying the most effective antibody protocols from literature, pre-prints, and patents, allowing researchers to streamline their experiments and accelerate their discoveries.

Most cited protocols related to «Fluorescent Antibody Technique»

1
Immunofluorescent Analysis of Ocular Proteins
Cited 68 times

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Publication 2021
Actins Animals Antibodies Biopharmaceuticals Capsule Catabolism Cataract Cells DNA Replication Epithelium Epitopes Eye Fibrosis Fluorescent Antibody Technique Fluorescent Dyes Freezing Gene Expression Genes Homo sapiens Immunoglobulins Institutional Animal Care and Use Committees Lens, Crystalline Mice, Inbred C57BL Microscopy, Confocal Mus Operative Surgical Procedures Protein Denaturation Proteins RNA-Seq Smooth Muscles Tissues Training Programs Vision Western Blotting
2
Cardiomyocyte Ablation and Lineage Tracing in Zebrafish
Cited 48 times
Chamber-specific ablation and reporter lines were generated using the standard I-SceI meganuclease transgenesis technique (details in Methods). To perform ventricular cardiomyocyte ablation, Tg(vmhc:mCherry-NTR) zebrafish were treated with 5 mM MTZ as previously described9 (link). For lineage tracing experiments, Tg(vmhc:mCherry-NTR;amhc:CreERT2;β-act2:RSG) zebrafish were treated with 10 µM 4-hydroxytamoxifen as previously described5 (link). For Notch inhibition studies, zebrafish were treated with 100 µM DAPT. Live imaging, heart contraction, immunofluorescence, and whole mount in situ hybridization were performed as described in Methods.
Zhang R., Han P., Yang H., Ouyang K., Lee D., Lin Y.F., Ocorr K., Kang G., Chen J., Stainier D.Y., Yelon D, & Chi N.C. (2013). In Vivo Cardiac Reprogramming Contributes to Zebrafish Heart Regeneration. Nature, 498(7455), 497-501.
Publication 2013
1,2-dilinolenoyl-3-(4-aminobutyryl)propane-1,2,3-triol CCL4 protein, human Fluorescent Antibody Technique Heart Ventricle hydroxytamoxifen In Situ Hybridization Myocardial Contraction Myocytes, Cardiac Psychological Inhibition Zebrafish
3
Single-Molecule Immunofluorescence Imaging
Cited 46 times
Flow chambers were prepared on mPEG passivated quartz slides doped with biotin PEG15 (link). Biotinylated antibodies were immobilized by incubating ~10 nM of antibody for 10 min on NeutrAvidin (Thermo) coated flow chambers. Prism type total internal reflection fluorescence (TIRF) microscope was used to acquire the single molecule data40 (link). Samples with fluorescent protein tag were serially diluted to obtain well-isolated spots on the surface upon 20 min of incubation over immobilized antibody surface. All dilutions were made immediately before addition to the flow chamber in 10 mM Tris-HCl pH 8.0, 50 mM NaCl buffer with 0.1 mg/ml bovine serum albumin (New England Biolabs), unless specified. Unbound antibodies and sample were removed from the channel by washing with buffer twice between successive additions. For immunofluorescence detection, immunoprecipitated complexes were incubated with a different antibody against prey protein (~10 nM) for 20 min and fluorescent-dye-labeled secondary antibody (2–5 nM) for 5 min before imaging. Single molecule analysis was performed using scripts written in Matlab.
Jain A., Liu R., Ramani B., Arauz E., Ishitsuka Y., Ragunathan K., Park J., Chen J., Xiang Y.K, & Ha T. (2011). Probing Cellular Protein Complexes via Single Molecule Pull-down. Nature, 473(7348), 484-488.
Publication 2011
Antibodies Biotin Buffers Exanthema Fluorescent Antibody Technique Fluorescent Dyes Immunoglobulins Microscopy, Fluorescence monomethoxypolyethylene glycol neutravidin prisma Proteins Quartz Reflex Serum Albumin, Bovine Single Molecule Analysis Sodium Chloride Technique, Dilution Tromethamine
4
Single-Molecule Immunofluorescence Imaging
Cited 46 times
Flow chambers were prepared on mPEG passivated quartz slides doped with biotin PEG15 (link). Biotinylated antibodies were immobilized by incubating ~10 nM of antibody for 10 min on NeutrAvidin (Thermo) coated flow chambers. Prism type total internal reflection fluorescence (TIRF) microscope was used to acquire the single molecule data40 (link). Samples with fluorescent protein tag were serially diluted to obtain well-isolated spots on the surface upon 20 min of incubation over immobilized antibody surface. All dilutions were made immediately before addition to the flow chamber in 10 mM Tris-HCl pH 8.0, 50 mM NaCl buffer with 0.1 mg/ml bovine serum albumin (New England Biolabs), unless specified. Unbound antibodies and sample were removed from the channel by washing with buffer twice between successive additions. For immunofluorescence detection, immunoprecipitated complexes were incubated with a different antibody against prey protein (~10 nM) for 20 min and fluorescent-dye-labeled secondary antibody (2–5 nM) for 5 min before imaging. Single molecule analysis was performed using scripts written in Matlab.
Jain A., Liu R., Ramani B., Arauz E., Ishitsuka Y., Ragunathan K., Park J., Chen J., Xiang Y.K, & Ha T. (2011). Probing Cellular Protein Complexes via Single Molecule Pull-down. Nature, 473(7348), 484-488.
Publication 2011
Antibodies Biotin Buffers Exanthema Fluorescent Antibody Technique Fluorescent Dyes Immunoglobulins Microscopy, Fluorescence monomethoxypolyethylene glycol neutravidin prisma Proteins Quartz Reflex Serum Albumin, Bovine Single Molecule Analysis Sodium Chloride Technique, Dilution Tromethamine
5
Immunofluorescence Staining of XFP-Labeled Neurons
Cited 45 times
Animals were perfused with 4% paraformaldehyde in 0.1 M PB. Brains were fixed for an additional 18 hr at 4°C. Brains were then cryoprotected in 20% (w/v) sucrose in PBS at 4°C overnight. Brains were embedded in OCT and 25-μm cryosections were cut using the tape transfer method. For direct imaging of XFP, sections were DAPI stained and coverslipped as above. For single-label immunofluorescence, sections were incubated for 30 min at room temperature in PBS containing 0.3% Triton X-100 and 5% normal goat serum (NGS). Sections were incubated with anti- GFP (Abcam; 1:1000 dilution) overnight at 4°C. Sections were rinsed for 30 min in PBS containing 1% NGS, incubated in goat anti-rabbit IgG-Alexa Fluor 488 (Invitrogen; 1:400 dilution) for 2 hr at room temperature, rinsed for 10 min in PBS containing 1% NGS, and rinsed for 30 min in PBS. Sections were then DAPI stained and coverslipped as above. XFP or IHC imaging was identical to the DFISH automated fluorescence microscopy method.
Madisen L., Zwingman T.A., Sunkin S.M., Oh S.W., Zariwala H.A., Gu H., Ng L.L., Palmiter R.D., Hawrylycz M.J., Jones A.R., Lein E.S, & Zeng H. (2009). A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nature neuroscience, 13(1), 133-140.
Publication 2009
alexa fluor 488 Animals anti-IgG Brain Cryoultramicrotomy DAPI Fluorescent Antibody Technique Goat Microscopy, Fluorescence paraform Rabbits Serum Sucrose Technique, Dilution Triton X-100

Most recents protocols related to «Fluorescent Antibody Technique»

1
Antibody Cytotoxicity Assay in AQP4-GFP Cells
Not available on PMC !

Example 4

HEK293-hAQP4-GFP and LPS-stimulated RAW264.7 were co-cultured with CD4 antibody, commercial AQP4 antibody, A002 antibody (ten-fold serial dilution) or culture medium only at 37° C. and 5% CO 2 for 6 hours. Cells were stain with Fixable Far Red-labeled anti-amine, PE-labeled anti-mouse CD11 b then analyzed the % of amine in CD11b-/GFP+ cells. Cell death(%) increased in ADCC=(% cell death in presence of IgG-% cell death in absence of IgG)/(% Cell death in maximum lysis-% cell death in absence of IgG)×100. Antibody-dependent cell-mediated cytotoxicity Assay (ADCC) of A002 antibody by immunofluorescent stain. HEK293-hAQP4-GFP and LPS-stimulated RAW264.7 were co-cultured with CD4 antibody, commercial AQP4 antibody, A002 antibody (ten-fold serial dilution) or culture medium only at 37° C. and 5% CO2 for 6 hours. Then cells were stain with Propidium Iodide (PI). Histograms show quantification of Propidium Iodide (PI) of cells co-cultured with CD4 antibody, commercial AQP4 antibody or A002 antibody (ten-fold serial dilution) (FIG. 9). PI intensity was adjusted by Subtracting PI intensity of cells co-cultured with culture medium only.

US11858989B2. Anti-AQP4 antibodies (2024-01-02). None [TW]. Inventors: Chao-Lin Lee [TW].
Full text: Click here
Patent 2024
Amines Biological Assay Cell Death Cells Cytotoxicities, Antibody-Dependent Cell Fluorescent Antibody Technique Immunoglobulins ITGAM protein, human Mus Propidium Iodide Stains Technique, Dilution
2
Quantifying Checkpoint Inhibitor Expression
Not available on PMC !

Example 2

Experiments to determine expression levels of checkpoint inhibitors: PD-1 and LAG-3 on cells in the experiments described below used the following appropriately fluorescent labeled commercial antibodies (phycoerythrin-cyanine7 (PE-Cy7)-conjugated anti-CD4 [clone SK3] or fluorescein isothiocyanate (FITC)-conjugated anti-CD4 [clone RPA-T4], phycoerythrin (PE)-conjugated anti-LAG-3 [clone 3DS223H], phycoerythrin (PE)-conjugated anti-PD-1 [clone EH12.2H7] or allophycocyanin (APC)-conjugated (eBiosciences, or BioLegend)) and the appropriate isotype controls. All antibodies were used at the manufacturer's recommended concentrations. Cell staining was performed in FACS buffer (10% FCS in PBS) on ice for 30 minutes in the dark for the addition of primary antibodies. After two washes, cells were either stained with the appropriate secondary reagent on ice for 30 minutes in the dark or immediately analyzed on a flow cytometer. To exclude dead cells, all samples were co-stained with a viability dye: 7-Aminoactinomycin D (7-AAD) (BD Biosceinces, or BioLegend) or 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) (Life Technologies). All samples were analyzed on either a FACS Calibur or Fortessa Flow Cytometer (BD Biosciences) and analyzed using FlowJo Software (TreeStar, Ashland, Oreg.).

US11858991B2. LAG-3-binding molecules and methods of use thereof (2024-01-02). MacroGenics, Inc. [US]. Inventors: Ross La Motte-Mohs [US], Kalpana Shah [US], Douglas H. Smith [US], Leslie S. Johnson [US], Paul A. Moore [US], Ezio Bonvini [US], Scott Koenig [US].
Full text: Click here
Patent 2024
7-aminoactinomycin D allophycocyanin Antibodies Buffers Cell Cycle Checkpoints Cells Clone Cells DAPI Flow Cytometry Fluorescein Fluorescent Antibody Technique Immunoglobulin Isotypes inhibitors isothiocyanate Phycoerythrin
3
Immunofluorescence Staining of Rat Cornea
The eyeballs of rats were fixed with 4% paraformaldehyde for 2 h, then embedded with optimal cutting temperature compound (OCT) at –80 °C and cut into 8-μm-thick sections for their immunofluorescence staining. Cryosections were washed twice with PBS, permeabilized with 0.3% Triton X-100 in PBS, and blocked in 0.1% Triton X-100 and 2% bovine serum albumin (BSA) in PBS. The blocked corneal tissue was then incubated overnight at 4 °C with primary antibodies: these were p38, phospho-p38 MAPK (Thr180/Tyr182), ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), JNK, and p-JNK (Thr183/Tyr185) (Cell Signaling Technology; Danvers, MA, USA). After the corneal cells were incubated with secondary antibodies, their nuclei were counterstained with DAPI. All sections were detected using CLSM (LSM 800, Zeiss, Germany).
Liu J., Yang S., Zhao L., Jiang F., Sun J., Peng S., Zhao R., Huang Y., Fu X., Luo R., Jiang Y., Li Z., Wang N., Fang T, & Zhang Z. (2023). ROS generation and p-38 activation contribute to montmorillonite-induced corneal toxicity in vitro and in vivo. Particle and Fibre Toxicology, 20, 8.
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Publication 2023
Antibodies Bos taurus Cell Nucleus Cells Cornea Cryoultramicrotomy DAPI Eye Fluorescent Antibody Technique Mitogen-Activated Protein Kinase 3 Mitogen-Activated Protein Kinase p38 paraform Rattus Serum Albumin Tissues Triton X-100
4
Maternal Vaping Impacts Offspring Neurobehavior
All studies were approved by the IACUC of Texas Tech University Health Sciences Center, Lubbock, Texas (IACUC protocol# 20026). Experiments were performed in accordance with relevant guidelines and regulations. Female CD1 pregnant mice (Charles River Laboratories, Inc., Wilmington, MA; Cat# CRL: 22, RRID: IMSR_CRL:22) and after delivery their offspring were kept under standardized light and dark conditions (12 h), humidity (70%), and temperature (22 °C). Pregnant mice were singly housed. Offspring were separated into male and female after weaning (postnatal day 21–22) and housed in a group of 2–5. They were given ad libitum access to food and water. Animal behavior was monitored daily to minimize animal suffering. We applied the following exclusion criteria to our experiments: severe weight loss, infections, or significant behavioral deficits (decreased mobility, seizures, lethargy). No animal was excluded from this study. The research design is depicted in Fig. 1. A total number of 176 (n = 16 for mother and n = 160 for offspring) mice were used to perform this study. All experiments were conducted in compliance with the ARRIVE guidelines.

Study design. Pregnant CD1 were exposed to Blu e-cigarette from gestational day 5 (E5) to postnatal day 7 (PD7). At the end of the exposure, plasma nicotine and cotinine level were measured by LCMS/MS, and body weight was measured at PD7, PD23, PD45 and PD90. Mice were sacrificed and brain was extracted at every time point to evaluate blood-brain barrier (BBB) integrity by western blot and immunofluorescence. Open field test, novel object recognition test and morris water maze test were conducted at adolescent and adult time point to evaluate hyperactivity and learning-memory function

Archie S.R., Sifat A.E., Zhang Y., Villalba H., Sharma S., Nozohouri S, & Abbruscato T.J. (2023). Maternal e-cigarette use can disrupt postnatal blood-brain barrier (BBB) integrity and deteriorates motor, learning and memory function: influence of sex and age. Fluids and Barriers of the CNS, 20, 17.
Full text: Click here
Publication 2023
Adult Animals Body Weight Brain Cotinine Females Fluorescent Antibody Technique Food Humidity Infection Institutional Animal Care and Use Committees Laser Capture Microdissection Lethargy Light Males Memory Mice, House Morris Water Maze Test Mothers Nicotine Novel Object Recognition Test Obstetric Delivery Open Field Test Plasma Pregnancy Range of Motion, Articular Rivers Seizures Western Blot
5
Immunofluorescence Staining of Brain Tissue
Immunofluorescence staining was performed as previously described with modifications [45 (link), 46 (link)]. Mice were euthanized by isoflurane overdose at each time point. The brains were sectioned at 20 µM of thickness, fixed with 4% paraformaldehyde (Thermo Fisher) for 15 min, then permeabilized with 0.1% Triton X-100 for 10 min. After washing with the phosphate-buffered saline (PBS) for 15 min, the sections were blocked for 1 h and incubated overnight with primary antibodies for ZO-1 (1:100, Thermo Fisher) claudin-5 (1: 100, Thermo Fisher) and GFAP (1:100, Cell Signaling), respectively. Alexa fluorescent secondary antibodies (Thermo Fisher) were used at 1:400 dilutions for 1 h. After counterstaining with 4′,6-diamidino-2-phenylindole (DAPI) for nucleus and washing with PBS, the sections were mounted with Permount (Thermo Fisher). The whole sections were scanned with a Leica Stellaris SP8 Falcon microscope (Leica Microsystem) and the images (20X magnitude) were captured with the same microscope. Mean total fluorescence intensity was calculated for each color channel and intensity of green color (ZO-1/GFAP) and red color (claudin-5) was expressed relative to blue color (DAPI). Cortex and hippocampus of both hemispheres of each brain section were used to evaluate the expression levels of ZO-1, claudin-5 and GFAP. To minimize the subjective bias, all images for ZO-1, claudin-5 and GFAP expression analysis were captured under the same microscopic parameter (laser power, pinhole size, exposure time) setting.
Archie S.R., Sifat A.E., Zhang Y., Villalba H., Sharma S., Nozohouri S, & Abbruscato T.J. (2023). Maternal e-cigarette use can disrupt postnatal blood-brain barrier (BBB) integrity and deteriorates motor, learning and memory function: influence of sex and age. Fluids and Barriers of the CNS, 20, 17.
Full text: Click here
Publication 2023
Antibodies Brain Cell Nucleus Cerebral Hemispheres Claudin-5 Cortex, Cerebral Drug Overdose Fluorescence Fluorescent Antibody Technique Glial Fibrillary Acidic Protein Isoflurane Microscopy MLL protein, human Mus paraform Phosphates Saline Solution Seahorses Technique, Dilution Triton X-100

Top products related to «Fluorescent Antibody Technique»

1
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DAPI is a fluorescent dye that binds strongly to adenine-thymine (A-T) rich regions in DNA. It is commonly used as a nuclear counterstain in fluorescence microscopy to visualize and locate cell nuclei.
2
4 6 diamidino 2 phenylindole (dapi) by Thermo Fisher Scientific
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3
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Alexa Fluor 488 is a fluorescent dye used in various biotechnological applications. It has an excitation maximum at 495 nm and an emission maximum at 519 nm, producing a green fluorescent signal. Alexa Fluor 488 is known for its brightness, photostability, and pH-insensitivity, making it a popular choice for labeling biomolecules in biological research.
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Bovine serum albumin (BSA) is a common laboratory reagent derived from bovine blood plasma. It is a protein that serves as a stabilizer and blocking agent in various biochemical and immunological applications. BSA is widely used to maintain the activity and solubility of enzymes, proteins, and other biomolecules in experimental settings.
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Hoechst 33342 by Thermo Fisher Scientific
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Lsm 710 by Zeiss
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The LSM 710 is a laser scanning microscope developed by Zeiss. It is designed for high-resolution imaging and analysis of biological and materials samples. The LSM 710 utilizes a laser excitation source and a scanning system to capture detailed images of specimens at the microscopic level. The specific capabilities and technical details of the LSM 710 are not provided in this response to maintain an unbiased and factual approach.
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DAPI is a fluorescent dye used for staining and visualizing DNA in biological samples. It binds to the minor groove of DNA, emitting blue fluorescence when excited by ultraviolet or violet light. DAPI is commonly used in fluorescence microscopy and flow cytometry applications.

More about "Fluorescent Antibody Technique"

The Fluorescent Antibody Technique (FAT) is a powerful analytical method that utilizes fluorescently labeled antibodies to detect and localize specific target molecules within biological samples.
This technique, also known as immunofluorescence or immunocytochemistry, allows researchers to visualize the distribution and abundance of proteins, cells, or other biomolecules of interest, making it a valuable tool in a wide range of applications, such as immunohistochemistry, flow cytometry, and fluorescence microscopy.
By leveraging the specificity of antibody-antigen interactions and the sensitivity of fluorescent detection, the FAT enables researchers to gain valuable insights into the complex biological processes underlying health and disease.
Commonly used fluorescent dyes and labels in this technique include DAPI (4',6-diamidino-2-phenylindole) for nuclear staining, Alexa Fluor 488 for green fluorescence, and Hoechst 33342 for counterstaining.
To optimize the FAT, researchers often utilize detergents like Triton X-100 for permeabilization and blocking agents like bovine serum albumin (BSA) to reduce non-specific binding.
The resulting fluorescent signals can then be captured and analyzed using advanced fluorescence microscopy techniques, such as confocal laser scanning microscopy (e.g., LSM 710) or epifluorescence microscopy.
PubCompare.ai's AI-driven protocol comparison tool can help streamline the FAT process by identifying the most effective antibody protocols from literature, pre-prints, and patents.
By comparing multiple protocols side-by-side, researchers can quickly identify the optimal conditions and reagents for their specific experiments, accelerating their discoveries and advancing their understanding of complex biological systems.