Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
Prevention of fluorescence fading
What is available in the way of chemical additives to aqueous mounting media, commercial or homemade, to suppress fading of immunofluorescence preparations?
Jules Elias has a discussion about this in his book "Immunohistopathology, A practical approach to diagnosis." ASCP Press, 1990. He says 1 percent p-phenylenediamine
added to the mounting medium retards fading.
Two references he gives:
Johnson, GD, et al, A Simple Method of Reducing the Fading of Immunofluorescece During Microscopy. J Immunol Methods 43:349-380, 1981.
Huff, JC, et.al., Enhancement of Specific Immunofluorescent Findings with use of para-phenylenediamine mounting
buffer. J Invest Dermatol 78:49, 1982.
Look into Vectashield, it is supposed to a good mounting media for immunofluorescence. You may not be able to prevent fading entirely, because the exciting light can cause it. Storage of the slides, after coverslipping, should be dark, sometimes in cold, or even in a freezer.
Vectashield is from Vector and it is pricey: $40 for 10 ml.
I think that the anti-fade agents that have already been mentioned are all good, I must admit I have never used Vectashield so will not comment on this. However, no mention has been made of the possible variability in results with these materials. Most of the anti-fade agents I have tried vary considerably in their effectiveness. This appears to depend on the specific antibody used, the fluorescent marker, the fluorescence ratio of dye to marker molecule, whether the IHC is direct or indirect and if you remembered to feed your cat before going to work. As an example using lectin labelling of cells with direct or indirect techniques, I found that the FITC label was usually retained for UEA-1 but not for WGA. I would therefore urge anyone who is going to use anti-fade agents to try them first on some extra slides to test their effectiveness.