Immunofluorescence Information

In this section we feature our educational assets for using immunofluorescence in basic research. We have an extensive array of webinars, videos, application notes, articles and more to help you design and execute your experiments with confidence.

Immunofluorescence features

Selection guide

Guide to choosing a labeling strategy

This simple guide helps you decide which detection method is best suited for your experiment.

On-demand webinar

Five steps for publication-quality fixed-cell imaging the first time

With over 40 years dedicated to cell imaging research, we offer the five steps to help you confidently create quality fixed cell images the first time. Presented by Jason Kilgore, technical applications scientist at Thermo Fisher Scientific.


Immunofluorescence learning resources

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Type Title Categories
Application note (2013) Optimizing the high content analysis of cell morphology using multi-dimensional imaging on the Thermo Scientific ArrayScan XTI HCA Reader: Stem cell colonies  Alexa Fluor, antibodies, ArrayScan, confocal microscopy, fluorescence microscopy/fluorescence imaging, fluorescent dyes, high content analysis, immunofluorescence (IF), microplate reader, particles, stem cell research
Application note (2017) Flow cytometry analysis of transcription factor expression during differentiation of hPSC-derived cardiomyocytes antibodies, Attune/Attune NxT, cardiomyocytes, cell differentiation, flow cytometer/flow cytometry, fluorescence, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), immunophenotyping, stem cell research
BioProbes article (2006) A guide to antibody labeling and detection  Alexa Fluor, antibody labeling, flow cytometer/flow cytometry, fluorescent dyes, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), multicolor flow cytometry, nanocrystals, Qdot, Zenon
BioProbes article (2007) Looking for apoptosis?  antibodies, apoptosis, colorimetric, ELISA, flow cytometer/flow cytometry,fluorescent dyes, immunocytochemistry (ICC), protein detection, western blotting
BioProbes article (2008) Nuclear receptor antibodies: A core technology  antibodies, cell signaling, ELISA, gene expression, immunocytochemistry (ICC), immunofluorescence (IF), immunohistochemistry (IHC), ion flux/ion channels, western blotting
BioProbes article (2008) Fluorescent protein–based Organelle Lights and new Cellular Lights BacMam reagents  BacMam technology, cell structure-all, cell tracking, Cellular Lights, fluorescent proteins, fluorescence microscopy/fluorescence imaging, immunocytochemistry (ICC)
BioProbes article (2008) IgG antibody labeling from A to Z—Alexa Fluor to Zenon IgG Antibody Labeling Kits for imaging and flow cytometry  Alexa Fluor, antibody labeling, flow cytometer/flow cytometry, fluorescent dyes, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), protein labeling, Zenon
BioProbes article (2008) Analyzing signaling events with phosphorylation site–specific antibodies—Insights into functional proteomics  antibodies, antibody labeling, cell signaling, fluorescent dyes, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), protein labeling
BioProbes article (2009) Novel recombinant monoclonal antibodies—ABfinity technology ensures performance  antibodies, cell signaling, flow cytometer/flow cytometry, fluorescence microscopy/fluorescence imaging, immunohistochemistry (IHC), protein quantitation, sample preparation, western blotting
BioProbes article (2009) Baculoviral transduction of human neurons  chemiluminescence, ELISA,fluorescent dyes, gene expression, immunocytochemistry (ICC), immunofluorescence (IF), immunohistochemistry (IHC), neuroscience, stem cell research, western blotting
BioProbes article (2009) Fluorescence-based alternatives to chromogenic immunohistochemistry  antifades, cancer, fluorescent dyes, fluorescence microscopy/fluorescence imaging, immunohistochemistry (IHC), particles, protein quantitation, sample stability
BioProbes article (2009) Highly specific antibodies for cell junction studies  Alexa Fluor, antibodies, cell junction studies, ELISA, fluorescent dyes, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), particles, western blotting
BioProbes article (2009) Specific detection of gap junction proteins  Alexa Fluor, antibodies, fluorescent dyes, fluorescence microscopy/fluorescence imaging, immunocytochemistry (ICC), immunofluorescence (IF), immunohistochemistry (IHC), protein detection
BioProbes article (2009) Is it time to go direct? IgG antibody labeling methods as versatile as your imagination  Alexa Fluor, antibody labeling, flow cytometer/flow cytometry, fluorescent dyes, immunocytochemistry (ICC), immunohistochemistry (IHC), nanocrystals, Qdot, sample preparation, Zenon
BioProbes article (2011) New simplified workflow options for maximum-yield protein conjugations—From APEX to Zenon: Molecular Probes protein labeling kits Alexa Fluor, antibodies, antibody labeling, flow cytometry/flow cytometer, immunofluorescence (IF), protein labeling, Zenon
BioProbes article (2011) Focusing on the FAK signaling pathway—Phosphorylation-specific antibodies for Focal Adhesion Kinase (FAK) antibodies, cell signaling, fluorescence microscopy/fluorescence imaging, immunocytochemistry (ICC)
BioProbes article (2013) Beyond Lights Limits: Fluorescence imaging at the nanoscale—Fluorescent probes for three super-resolution modalities antibodies, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, fluorescent dyes, SIM, STORM, STED, super resolution microscopy
BioProbes article (2015) Applications for therapeutic monoclonal antibodies—Tools for studying antibody internalization and trafficking antibodies, antibody-drug conjugates, antibody labeling, drug research, endocytosis/phagocytosis, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), immunohistochemistry (IHC), internalization, pH detection
BioProbes article (2016) Histological analysis using the CellInsight CX7 HCA Platform—Quantitative imaging of histological samples CellInsight, high content analysis, immunocytochemistry (ICC), immunofluorescence (IF), immunohistochemistry (IHC)
BioProbes article (2016) Antibodies for pluriptent stem cells and neural lineage cells—Light up neural diffentiation pathways antibodies, high content analysis, immunocytochemistry (ICC), immunofluorescence (IF), immunohistochemistry (IHC), immunophenotyping, neuroscience, stem cell research
BioProbes article (2017) Boost your stem cell antibody arsenal—Monoclonal antibodies for studying human pluripotent stem cells antibodies, immunofluorescence (IF), pluripotent stem cells, stem cell research
BioProbes article (2017) Appetite for destruction—Studying macroautophagy with Premo fluorescent protein–based sensors autophagy, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, fluorescent dyes, fluorescent proteins, immunocytochemistry (ICC), immunofluorescence (IF), live-cell imaging
BioProbes article (2017) Effectively monitor antibody internalization and trafficking—Improved labeling tools for antibody–drug conjugate screening and characterization antibodies, antibody-drug conjugates, antibody labeling, drug research, endocytosis/phagocytosis, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), immunohistochemistry (IHC), internalization, pH detection
BioProbes article (2017) Amplify signals for fluorescence imaging—SuperBoost kits with Alexa Fluor tyramides for signal amplification Alexa Fluor, antibody labeling, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), secondary detection, signal amplification
BioProbes article (2017) Harness immune checkpoints to combat tumors—Immune checkpoint antibodies for flow cytometry, IHC, and functional bioassays antibodies, flow cytometer/flow cytometry, immunofluorescence (IF), immunohistochemistry (IHC), immunology, immuno-oncology
Molecular Probes Handbook TSA and other peroxidase-based signal amplification techniques—Section 6.2 Alexa Fluor, antibodies, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), reagents, secondary detection, signal amplification
Protocol Immunofluorescent staining of intracellular antigens on cultured cells fluorescence microscopy/fluorescence imaging, general ab staining, imaging, immunofluorescence (IF)
Scientific poster (2008) Novel Click-IT TUNEL Assay for detection of cell death  Alexa Fluor, antibodies, apoptosis, Click-iT, cytotoxicity, fluorescence microscopy/fluorescence imaging, fluorescent dyes, gel electrophoresis, high content analysis, immunocytochemistry (ICC), viability
Scientific poster (2010) Antibody- and fluorescent protein-based approaches to measuring autophagy in mammalian cells by fluorescence microscopy  Alexa Fluor, antibodies, autophagy, BacMam technology, cell proliferation, Click-iT, fluorescence microscopy/fluorescence imaging, fluorescent dyes, high content analysis, live-cell imaging
Scientific poster (2010) Proliferative and phenotypic characterization of human mesenchymal stem cells by flow cytometry and imaging  Alexa Fluor, cell proliferation, Click-iT, flow cytometer/flow cytometry, fluorescence microscopy/fluorescence imaging, fluorescent dyes, immunofluorescence (IF), immunophenotyping, stem cell research, violet laser-excited reagents
Scientific poster (2010) ABfinity antibodies: A novel approach to antibody development  Alexa Fluor, antibodies, flow cytometer/flow cytometry, fluorescence microscopy/fluorescence imaging, fluorescent dyes, gene expression, immunocytochemistry (ICC), western blotting
Scientific poster (2010) Identification and characterization of O-GlcNAc modification of galectin-1 in mesenchymal stem cells using click chemistry  Alexa Fluor, BacMam technology, Click-iT, fluorescence microscopy/fluorescence imaging, fluorescent dyes, gel electrophoresis, immunocytochemistry (ICC), protein enrichment, protein labeling, western blotting
Scientific poster (2015) Colorimetric dual labeled EdU/BrdU technology demonstrates contextual information and dynamics of proliferation in tissue  apoptosis, brightfield microscopy, cell proliferation, Click-iT, brightfield microscopy, fluorescence microscopy/fluorescence imaging, immunohistochemistry (IHC)
Scientific poster (2016) Correlating internalization and potency to accelerate antibody discovery and development brightfield microscopy, colorimetric, endocytosis and phagocytosis, EVOS,fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), immunohistochemistry (IHC)
Tutorial 1.2 Fixation–Fixed cell imaging: 5 steps for publication-quality images 
The next step of tissue preparation is fixation. Fixation refers to a chemical means of killing and preserving cells in a particular physiological state, and in many cases, to preserve morphology. Proper fixation equals preservation of target.
fixation, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF)
Tutorial 4.1 Mounting media–Fixed cell imaging: 5 steps for publication-quality images 
Using the right mounting media can impact your experiment. Be sure to choose the right type of mountant for your set-up.
antifade mounting media, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), mounting media
Tutorial 3.2 Secondary antibody optimization–Fixed cell imaging: 5 steps for publication-quality images 
Secondary antibody detection protocols also need to be optimized for each primary antibody used.
antibody labeling, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), secondary antibodies, secondary antibody protocol, secondary detection
Tutorial 4.2 Photobleaching and antifades–Fixed cell imaging: 5 steps for publication-quality images 
What is photobleaching and how can you prevent it from destroying your sample? Options for antifades are discussed.
antifades, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), photobleaching, Prolong Gold, Vectashield
Tutorial 5.5 Ethical considerations–Fixed cell imaging: 5 steps for publication-quality images 
Ethical imaging means trustworthy data, and thus, publishable data. How to treat your samples and data to preserve data integrity is presented.
ethical considerations, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, image analysis, immunofluorescence (IF), sample manipulation
Tutorial 5.2 Imaging platforms-software–Fixed cell imaging: 5 steps for publication-quality images 
Taking images on a microscope usually entails having some type of imaging software that aids in taking the image and assists in combining differing colors into one. There are some very important aspects to keep in mind to get a publishable image.
FiFI software, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, ImageJ, imaging software, imaging systems, immunofluorescence (IF)
Tutorial 1.1 Culture conditions–Fixed cell imaging: 5 steps for publication-quality images 
The first step in obtaining a good image is tissue preparation. For cultured cells, the cells must have good cell health and morphology, as well as good confluency. Healthy cells equal healthy data.
EVOS, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, imaging hardware, imaging systems, immunofluorescence (IF), microscope
Tutorial 1.5 Autofluorescence–Fixed cell imaging: 5 steps for publication-quality images 
The last step in cell preparation is autofluorescence. Cells and tissue can have a certain degree of autofluorescence that can confuse the specific signal, and lower the signal-to-background. Overcoming autofluorescence means greater sensitivity.
autofluorescence, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), tissue autofluorescence
Tutorial 5.4 Image analysis with Celleste software–Fixed cell imaging: 5 steps for publication-quality images 
The functionality of the Celleste software is reviewed and considerations for processing the image in different software programs are described.
Celleste, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, image analysis, immunofluorescence (IF)
Tutorial 1.4 Blocking–Fixed cell imaging: 5 steps for publication-quality images 
The next step after permeabilization is blocking, and there are a number of blocking techniques. Protein blocking equals specific antibody binding. Dye charge blocking means less non-specific binding.
dye charge blocking, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), nonspecific binding, protein blocking
Tutorial 2.2 Primary antibody protocol optimization–Fixed cell imaging: 5 steps for publication-quality images 
Every primary antibody must be optimized separately. There are many protocols available, and it is important to understand a "one size fits all" approach gives inferior results, as every antibody is slightly different. Learn how to approach optimization.
antibody labeling, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), primary antibodies, primary antibody protocol
Tutorial 5.1 Imaging platforms-hardware–Fixed cell imaging: 5 steps for publication-quality images 
The fifth step of the process is the actual imaging. To capture top-quality images, you need an imaging platform with top-of-the-line imaging capabilities. Here we review considerations for getting the best image.
EVOS, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, imaging hardware, imaging systems, immunofluorescence (IF), microscope
Tutorial 3.5 Dye choice and special concerns–Fixed cell imaging: 5 steps for publication-quality images 
There are many different dyes spanning the visible, far-red, and infrared wavelengths.Considerations for making the right choices for your experiment are presented.
dye choice, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, Fluorescence SpectraViewer, immunofluorescence (IF), label choice, muliparametric dye selection
Tutorial 1.3 Permeabilization–Fixed cell imaging: 5 steps for publication-quality images 
The next step is the permeabilization of the cells which is the key to opening intracellular compartments.
cell permeabilization, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF)
Tutorial 3.4 Controls–Fixed cell imaging: 5 steps for publication-quality images 
Researchers should conduct all necessary controls to rule out the possibility of non-specific binding or non-specific signal. Types of controls are described. Proper controls will boost your confidence in your final results.
fixed-cell imaging, fluorescence microscopy/fluorescence imaging, imaging controls, immunofluorescence (IF), sample controls
Tutorial 5.3 Image capture with EVOS FL Auto 2.0–Fixed cell imaging: 5 steps for publication-quality images 
Here the advantages of using the EVOS FL Auto 2.0 imaging system to capture your images are discussed.
EVOS FL Auto 2.0, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, image capture, immunofluorescence (IF)
Tutorial 3.3 Amplification techniques–Fixed cell imaging: 5 steps for publication-quality images 
If the signal is not strong enough using standard secondary detection schemes, you can increase the signal using amplification techniques. This is particularly important for low-expressing antigens, or rare-cell detection in samples. Examples are discussed.
biotin and streptavidin, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), signal amplification, tyramide detection
Tutorial 2.1 Primary antibody choice–Fixed cell imaging: 5 steps for publication-quality images 
After preparation, the second step to publishable images is to label the sample, usually involving primary antibodies to your specific targets of interest. The antibody source, the use of direct versus labeled antibodies as well as the validation for specific applications is discussed.
antibody labeling, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), primary antibodies
Tutorial 3.1 Secondary antibody choice–Fixed cell imaging: 5 steps for publication-quality images 
Step three of the five steps in making publishable images is to detect the label. That is, to detect with a secondary antibody, for instance, or an amplification technique, as well as to determine what controls to use. Your options are discussed.
antibody labeling, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), secondary antibodies, secondary antibody, secondary detection
Tutorial Labeling a purified antibody  
Educational video on how to label your next antibody for imaging or fluorescence. Walk through the tutorial as Molecular Probes scientists demonstrate the protocol—including all the tips and tricks you'll want to know about for your next antibody labeling experiment. The video features Judie showing Curtis, a chemistry graduate student, how to label his monoclonal antibodies while saving time and maximizing yield.
antibodies, antibody labeling, fluorescence microscopy/fluorescence imaging, fluorescent dyes, immunofluorescence (IF)
Video Tips and tricks: Phospho Immunofluorescence  cell analysis antibodies, cell imaging, fluorescence microscopy/fluorescence imaging, fixed-cell imaging, immunofluorescence (IF)
Video The secret to solving the most common problem in IF experiments  cell analysis antibodies, cell imaging, fluorescence microscopy/fluorescence imaging, fixed-cell imaging, immunofluorescence (IF)
Webinar Fixed cell imaging—Five steps for publication-quality images 
With over 40 years dedicated to cell imaging research, we offer long-proven tools and protocols to help confidently create quality cell images the first time. This on demand webinar covers the 5 essential steps to getting great images.
antibodies, blocking, Celleste, dyes, EVOS FL Auto 2.0, fixation, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF), sample detection, sample labeling, sample preparation, signal amplification
Webinar Learn to choose the right fluorophore when designing experiments 
The choice of fluorophore is one of the first important decisions to make in developing an experiment. Fluorophores are compounds that emit light at a specific wavelength when they have been excited at another, lower wavelength. Join our webinar and explore: How to choose the best organic dye for an assay Quantum dots and how they compare to other dyes When to use a phycobiliprotein like R-PE or APC When to use fluorescent proteins like GFP How to choose a suitable dye to match your instrument In addition, we will explore the basic characteristics, strengths, and weaknesses of the various fluorophores to help you choose and develop the best assay for your needs.
antibodies, antibody labeling, flow cytometer/flow cytometry, fluorescence microscopy/fluorescence imaging, fluorescent dyes, fluorescent proteins
Webinar A practical approach to antibody labeling 
The growing number of fluorophores available makes labeling your own antibodies a tempting proposition. But with many antibody labeling solutions available, selecting the best option can be a daunting task. In this webinar we will: Provide an overview of our antibody labeling kits Offer guidance on which methods are ideal for specific applications and experiments Provide tips and tricks to optimize your labeling protocol
antibodies, antibody labeling, flow cytometer/flow cytometry, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF)
Webinar An introduction to immunofluorescence staining of cultured cell 
In this webinar, we discuss the steps of an immunofluorescent staining protocol including material list, common variations, and necessary controls. We'll also provide a simple troubleshooting guide and examine how to avoid common pitfalls. Presented by Jason Kilgore, Technical Support Specialist, Thermo Fisher Scientific.
antibodies, antibody labeling,antifades, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunofluorescence (IF)
Webinar Basic techniques in autophagy research 
This webinar will introduce you to a series of analytical tools and techniques to help you identify and interrogate key features of autophagy. Topics to be covered include: Tips and tricks for selecting the right tools and achieving the best results Fluorescent proteins and antibodies used to analyze both live and fixed cells Analysis steps using a variety of multiplexing options, with quantitative methods for image analysis or fluorescence intensity measurement
autophagy, cell health, fluorescence microscopy/fluorescence imaging, fluorescent proteins, high content analysis, immunofluorescence (IF)
Webinar A comparison of basic immunofluorescent labeling strategies 
In this free webinar, we will compare different immunofluorescent labeling strategies exploring the pros and cons of each method. You will learn when the use of a direct conjugate is appropriate and when amplification techniques can be utilized. We'll also present a simple decision tree to aid in determining the best method for each situation.
Alexa Fluor, antibodies, antibody labeling, fixed-cell imaging, fluorescence microscopy/fluorescence imaging, immunocytochemistry (ICC), immunofluorescence (IF)