Comparison of antifading agents used in immunofluorescence

Various chemicals including p-phenylenediamine, n-propyl gallate, 1,4-diazobicyclo[2,2,2]-octane and Citifluor were tested to determine their effectiveness in retarding the fading of fluorescein-conjugated antibody bound to eosinophil granule major basic protein in tissues. Immunofluorescence fading and intensity were measured quantitatively using the Zeiss Zonax microphotometer system. All of these agents effectively retarded fluorescence fading; p-phenylenediamine and n-propyl gallate were the most effective. Comparison of glycerol-based and polyvinyl alcohol-based media containing n-propyl gallate or p-phenylenediamine showed that the glycerol-based medium yields higher mean fluorescence intensities than the polyvinyl alcohol-based medium. Slides mounted with p-phenylenediamine-glycerol could be stored for up to 2 weeks without loss of fluorescence intensity. Although p-phenylenediamine has many undesirable chemical properties, we conclude that it is the agent of choice for retarding the fading of tissue preparations stained with fluorescein isothiocyanate conjugates when used in a glycerol-based medium.