AAT Bioquest

What are some antifading agents used to prevent photobleaching?

Posted April 6, 2023


Antifade reagents are one of the main ingredients added to mounting mediums in fluorescence microscopy to reduce or prevent photobleaching of fluorescent dyes. Most antifade reagents are reactive oxygen species scavengers. Three of the most used anti-fading agents used to prevent photobleaching are p-Phenylenediamine (PPD), n-Propyl gallate (NPG), and 1,4-Diazabicyclo-octane (DABCOJ). 

p-Phenylenediamine (PPD) - One of the most effective anti-fading agents in mounting media, use of p-Phenylenediamine (PPD) results in diffused and weak fluorescence following storage of the stained slides. The downside is that it can react with cyanine dyes, splitting the cyanine molecule into two. 

n-Propyl gallate (NPG) – Another widely used antifade compound, n-Propyl gallate (NPG) is nontoxic and is commonly used with live cells. The downside is that it has anti-apoptotic properties, which could disrupt the biological process being studied. Also, it requires prolonged heating over several hours to dissolve.   

1,4-Diazabicyclo-octane (DABCOJ) – 1,4-Diazabicyclo-octane (DABCOJ) is comparatively less effective than p-Phenylenediamine but it is also less toxic. Although it is the preferred antifading agent for live-cell work, it is thought to shield against apoptosis, similar to n-Propyl gallate.

Additional resources

Comparison of anti-fading agents used in fluorescence microscopy: image analysis and laser confocal microscopy study

FluoroQuest™ PLUS Antifade Mounting Medium