Fixed-Cell Imaging Reagents
To expedite cell fixation and permeabilization, we’ve developed pre-packaged Molecular Probes® reagent kits with optimized workflows for common procedures including:
Find products for fixed-cell imaging
Proper fixation stops cellular metabolism and maintains protein structures by crosslinking. Inadequate fixation leads to cells being lost and disrupted by wash steps. Harsh fixation conditions can cause cellular disruption and create autofluorescent signals.
|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..|
Formaldehyde-fixed tissues and cells are impermeant to most antibodies—permeabilization is necessary to allow dyes and antibodies to penetrate. Treatment with detergent is the most common permeabilization method, and optimal permeabilization should both permit antibody and dye molecules to penetrate the cell interior and preserve cell-surface antigens.
|After fixation and permeabilization, U2OS cells were stained with NucBlue® Live Cell Stain, ActinRed™ 555 Ready Probes® Reagent, mouse anti–human golgin 97 primary, and goat anti-mouse IgG Alexa Fluor® 488 secondary. Treatment A used 0.05% Triton® X-100 for permeabilization. Treatment B used the Image-iT® Fixation/Permeabilization Kit, which provides optimal fixation conditions for most cell types. Treatment A showed lower staining intensity for the antibody-based secondary detection due to reduced membrane permeability.|
Dye charge blocking
Negatively charged dye molecules conjugated to antibodies or peptides will readily bind to positively charged structures. In cells, the dyes typically bind to mitochondia and nuclei, and in tissue like brain sections they bind to myelinated white matter, generating high background signals.
|U2OS cells were fixed using the Image-iT® Fixation/Permeabilization Kit. The ER was labeled using mouse anti-PDI and Alexa Fluor® 488 goat anti-mouse IgG (H+L) antibody, and nuclei were stained with NucBlue® Fixed Cell Stain. Treatments A and B both used standard blocking procedures to minimize non-specific protein interactions. Treatment B also used Image-iT® FX Signal Enhancer to reduce the charge-based non-specific binding of dye molecules to proteins. This non-specific binding can cause high background signals that obscure fine details in cell structure.|
The non-specific binding of antibodies to proteins other than the target antigen causes high background signals. Minimizing background from nonspecific binding of antibodies is essential to maximize signal-to-background ratios and improve sensitivity. This is especially important when looking for low-expressing antigens, using samples with high autofluorescence, or for techniques where signals are dimmer.
|U2OS cells were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit, then stained with NucBlue® Live Cell Stain, ActinRed™ 555 Ready Probes® Reagent, mouse anti–human golgin 97 primary and goat anti-mouse IgG Alexa Fluor® 488 secondary. Treatment A was not blocked; Treatment B includes BlockAid™ Blocking Solution. The non-blocked cells show a diffuse background staining from both nuclear stains (blue) and secondary antibodies (green), which obscures faint signals and limits quantitation. BlockAid™ Blocking Solution reduces the background signal resulting from non-specific protein interactions.|
Mammalian cells and tissues contain biotin-dependent carboxylases, which are required for a variety of metabolic functions. These biotin-containing enzymes often produce substantial background signals when biotin–streptavidin or biotin–avidin detection systems are used to identify cellular targets. Endogenous biotin is particularly prevalent in mitochondria and in kidney, liver, and brain tissues.
|A549 cells were fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit and labeled with a primary antibody against mouse anti-golgin-97 followed by DSB-X biotin-goat anti-mouse IgG, then stained with Alexa Fluor® 488 streptavidin, Alexa Fluor® 647 phalloidin, and NucBlue™ Fixed Cell Stain. After fixation, cells in B were treated with the Endogenous Biotin-Blocking Kit to reduce the background signal generated when biotin-avidin or biotin-streptavidin detection systems are used to identify cellular targets.|
Loss of fluorescence through irreversible photobleaching processes can lead to a significant reduction in sensitivity, particularly when target molecules are of low abundance or when excitation light is of high intensity or long duration.
To minimize photobleaching of experimental samples we have developed a series of antifade reagents which have been shown to increase the photostability of many popular fluorophores in a variety of sample types.
- Learn more about mounting medium and antifades
|A 60-second time-lapse showing the enhanced resistance to photobleaching afforded by ProLong® antifade reagents. Fixed HeLa cells were labeled with fluorescein phalloidin and mounted in ProLong® Diamond reagent, ProLong® Gold reagent, or 50% PBS/glycerol. Images were acquired at 12-second intervals using a 20x objective with continuous illumination from a standard 100-watt Hg-arc lamp.|