Improved fixed-cell imaging results

Pre-packaged reagent kits with optimized workflows combined with easy-to-use and powerful microscopes enable you to get the most from your fixed-cell imaging experiments. Below you can find products for fixation and permeabilization, cellular labeling, and image capture.

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5 steps for effective fixed-cell imaging

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:

Prepare your cells for fluorescent labeling.

 

Target cell structures and proteins of interest with organelle-selective dyes and stains and primary antibodies.

 

Fine-tune fluorescence signals for detailed observation.

 

Maintain photostability of fluorescence signals of samples.

 

Capture imaging discoveries with maximum clarity and definition.

 

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.

Stained cell images, with and without optimized fixation   After fixation and permeabilization, U2OS cells were stained with NucBlue™ Live Cell Stain and ActinGreen™ 488 Ready Probes™ Reagent. Treatment A used methanol-based solution for fixation, and Treatment B used the formaldehyde-based Image-iT™ Fixation/Permeabilization Kit. The methanol-based fixation in A results in fragmentation of the actin cytoskeleton and disruption of the cells. The Image-iT™ Fixation/Permeabilization Kit provides optimal fixation conditions for most cell types..

Product Description Cat. No.
Image-iT™ Fixation/Permeabilization Kit
  • Fixative: high-purity 4% formaldehyde in PBS, pH 7.3
  • Permeabilization solution: 0.5% Triton™ X-100
  • Blocking buffer: 3% BSA, fraction V, delipidated, New Zealand source, in DPBS
  • Wash solution: PBS, pH 7.4
R37602
BlockAid™ Blocking Solution 50 mL B10710

Label

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.

Cultured cells were prepared for staining using the Invitrogen™ Image-iT™ Fixation/Permeabilization Kit and were treated with Invitrogen™ BlockAid™ Blocking Solution. The sample was labeled with a primary antibody that recognizes mitochondria, followed by an Alexa Fluor 750 dye–conjugated secondary antibody (purple), Invitrogen™ NucBlue™ cell stain (blue), and Invitrogen™ ActinGreen™ 488 ReadyProbes™ Reagent (green). The image was captured on an Invitrogen™ EVOS™ FL Auto Imaging System.


Detect

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

Fixed and permeabilized HeLa cells, treated using the reagents in the Image-iT Fixation/Permeabilization Kit, were incubated with an anti-tubulin primary antibody and an Alexa Fluor 488 goat anti–mouse IgG (H+L) secondary antibody. Cells were then incubated with an anti–ATP synthase subunit IF1 antibody and labeled with the reagents in the Alexa Fluor 594 Tyramide SuperBoost Kit (goat anti–mouse IgG and Alexa Fluor 594 tyramide). Nuclei were labeled with NucBlue Fixed Cell ReadyProbes Reagent. Images were acquired on a confocal microscope.

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

Labeled Ex/Em Streptavidin Tyramide SuperBoost kits Goat anti–mouse IgG Goat anti–rabbit IgG
Goat anti–mouse IgG Goat anti–rabbit IgG
Alexa Fluor 488 495/519 S11223 B40912 B40922 A21121 A11070
Alexa Fluor 555 555/565 S21381 B40913 B40923 A21137 A21430
Alexa Fluor 594 591/617 S11227 B40915 B40925 A21044 A11072
Alexa Fluor 647 650/668 S21374 B40916 B40926 A21240 A21245
Biotin-XX B40911 B40921 A10519 B2770

Protect

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.

Explore Invitrogen™ ProLong™ antifades for fixed-cell imaging

A 60 sec time course shows the resistance to photobleaching achieved by Invitrogen™ ProLong™ Diamond Antifade Mountant. Fixed HeLa cells were labeled with Invitrogen™ FITC phalloidin and mounted in ProLong Diamond Antifade Mountant or 50% phosphate-buffered saline (PBS)/glycerol. Images were acquired at 12 sec intervals using a 20x objective with continuous illumination from a standard 100 W Hg-arc lamp.

 


Image

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 ›

Basic transmitted light-digital inverted system Advanced transmitted-light digital inverted system Basic fluorescence system Advanced fluorescence system Fully automated fluorescence system

EVOS XL Core
Perfect for cell culture and routine cell maintenance

EVOS XL
Perfect for more advanced colormetric assays

EVOS FLoid
Perfect for quick fluorescence visualization

EVOS FL
Perfect for multichannel fluorescence imaging and transfections

EVOS FL Auto
Perfect for a variety of advanced, automated applications