ProLong™ Live Antifade Reagent, for live cell imaging

Yes, this is possible.

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Yes. We recommend washing off the ProLong Live Antifade Reagent with pre-warmed buffer or media and proceeding to your standard fixation and permeabilization steps.

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This is possible for immediate viewing, but the sample cannot be archived with ProLong Live Antifade Reagent.

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As dyes are illuminated for imaging, they will fade, or “photobleach”, leading to unwanted dimming and lower detection efficiency over time. An antifade mounting medium can greatly reduce photobleaching. If you wish to label live cells, use of ProLong Live Antifade Reagent is helpful. If you wish to mount fixed cells after labeling, and then image immediately and then discard, SlowFade Diamond Antifade Mountant stays liquid but has good refractive index. If you wish to mount your sample and then archive the slides, ProLong Diamond Antifade Mountant will harden to a better refractive index and allow for archiving of the sample for up to several weeks, or even months. Unlike other antifade mounting media, these work well with fluorescent proteins for immediate viewing (archiving fluorescent proteins is not possible), and they are packaged with or without DAPI. More information on these can be found here (https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cellular-imaging/fluorescence-microscopy-and-immunofluorescence-if/mounting-medium-antifades/prolong-gold-antifade.html).

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

All fluorescent dyes will fade, or “photobleach”, to at least some extent when exposed to strong light at the wavelengths they absorb. Here are some causes for photobleaching and ways to fix the problem:

1) Cause of photobleacing - Generation of free radicals and singlet oxygen
Remedy - i) Use an antifade reagent, which has antioxidants and free radical scavengers:

ii) For live-cell imaging of fluorescent dyes and proteins, we recommend ProLong Live Antifade Reagent which can be added to the cell media or buffer. ProLong Live Antifade Reagent can significantly increase the stability over time for reagents as well as fluorescent proteins, like GFP, without affecting cell health, for up to 24 hours.

iii) For immediate analysis and short-term storage of fixed samples, we recommend SlowFade Diamond Antifade Mountant (it is a liquid mountant and can be used for immediate viewing and then disposal of the sample within a day).

iv) For long-term analysis of Alexa Fluor dyes in fixed samples, we recommend a mountant that hardens, such as ProLong Diamond Antifade Mountant. The harrdening of the mountant also slows diffusion of free radicals).

v) For long-term analysis of all dyes and fluorescent proteins in fixed samples, we recommend ProLong Diamond Antifade Mountant, suitable for archiving slides.

2) Cause of photobleaching - Dye is particularly sensitive to structural modification upon exposure to light.
Remedy - i) Choose a more photostable dye, such as many of our Alexa Fluor dyes.

3) Cause of photobleaching - Intense Illlumination
Remedy - i) Reduce light exposure, for example by reducing laser power or using neutral density filters.

ii) Minimize the viewing time of labeled sample, and close shutter when not viewing.

iii) Use an objective with a lower numerical aperture, such as a lower-power objective.

You can find more information on choosing an antifade reagent on the link below http://www.thermofisher.com/us/en/home/life-science/cell-analysis/cellular-imaging/fluorescence-microscopy-and-immunofluorescence-if/mounting-medium-antifades.html.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.