Follow these 5 steps to capture the best possible fixed-cell images to help ensure that your cell images are publication-ready the first time:
Fix, permeabilize, and block
To achieve optimal imaging quality, begin by setting up your study to spotlight proteins and cell structures of interest while keeping everything else out of the picture. Fixation and permeabilization prepare the cell samples for labeling—first by locking cellular structures, proteins, and nucleic acids in place, and then by making it possible for antibodies and fluorescent stains to permeate the interior of cells and label the targets of interest. Blocking prevents the fluorescent labels from nonspecifically binding to proteins that are not relevant to your research, thereby minimizing the signal-to-noise ratio.
Labeling various targets with separate fluorescent colors allows you to visualize different structures or proteins within cells in the same sample. Ways to label your target fluorescently include fluorescent dyes, immunolabeling, and fluorescent fusion proteins—all of which provide a means to selectively mark structures and proteins within the cell and allow you to see them more easily when you image.Many fluorescence tools for cell biology are essentially fluorophores that have been modified in different ways or conjugated to various molecules to give them a certain function or allow them to bind to specific organelles or proteins.Through chemical modifications, a single fluorophore can be produced in a number of variant forms, each with a different specificity. For example, the green-fluorescent Invitrogen Alexa Fluor 488 dye molecule can be modified to target actin filaments, can be attached to an IgG for use in immunolabeling, or can act as a whole-cell stain.The Invitrogen portfolio offers more than 51,000 high-quality primary antibodies. Some of these antibodies are attached directly to a broad range of intensely fluorescent markers and labels, including Invitrogen Alexa Fluor dyes. Explore our extensive portfolio of antibodies at thermofisher.com/primaryantibodies.
Detecting complex biological assemblies requires maximum clarity of fluorescence signals and separation of signals from background noise. Standard immunofluorescence labeling rarely provides the best signal-to-noise visibility. The difference between producing a good and a great publication-quality image requires fine-tuning your sample’s signal for peak specificity, definition, and amplification.
Quickly and easily choose the labeling solution you need
High- to medium-abundance protein targets
Secondary antibodies are used for the indirect detection of target antigens. While primary antibodies bind directly to the target, secondary antibodies bind indirectly by using the primary antibody as a bridge to the targeted biomolecule. This methodology serves to amplify signals and increase sensitivity to maximize detection.
Explore secondary antibodies
Medium- to low-abundance protein targets
Streptavidin conjugates can increase the number of fluorophores that label your target, and boost their signals. Streptavidin-based amplification techniques are widely used in fluorescence imaging for improved sensitivity of detection with primary and secondary antibodies.
Find out more about streptavidin signal amplification for imaging
Low-abundance protein targets
For low-abundance protein targets that are not detectable by conventional means, tyramide signal amplification (TSA, PerkinElmer) provides sensitive detection without compromising resolution. TSA technology employs an enzyme that releases reactive dyes in the presence of hydrogen peroxide to bring targets out of the background with definition and clarity.
Learn more about imaging low-abundance targets with TSA
Reagent Selection Table
Fluorophores are ideal for high-quality cell imaging but are inevitably prone to photobleaching, a photochemical degradation or fading of fluorescence signals. Any reduction in photosensitivity can skew your data and yield false results. Antifade mountants are designed to protect the photostability of fluorescently labeled proteins and maintain image integrity for weeks to months.
Key benefits of ProLong antifade mountants for fixed cells include:
- Inhibit photobleaching across the spectrum, even after prolonged storage
- Low background across the spectrum
- Hard-setting mountants
- Available with or without DAPI
- Ready-to-use benchtop formulations
- Choice of refractive index based on your needs: 1.52 for ProLong Glass reagent and 1.47 for ProLong Diamond and ProLong Gold reagents
Explore Invitrogen ProLong antifades for fixed-cell imaging
Capture research discoveries with maximum clarity and definition. In today’s competitive scientific environment, generating publication-quality images is critical to your success. To capture top-quality images, you need an imaging platform with top-of-the-line imaging components, including:
- High-quality cameras and optics to capture high-resolution images
- LED illumination to produce superior signal-to-noise ratios
- Easy-to-use image capture and processing software for ready-to-publish images
Compare the systems below to find the one that fits your imaging needs, or get more information on our entire line of EVOS Cell Imaging Systems ›