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Quenching Autofluorescence

When planning an immunofluorescent tissue stain, elements may autofluoresce causing problems interpreting the specific signal. 

Autofluorescence can be caused by a number of factors, one of the most common encountered is from formalin fixation of tissues, particularly problematic on archive material. There are also some tissue elements that also naturally fluoresce, including lipofuscin, collagen, elastin, and red blood cells.

There are a number of different solutions that tackle this issue, however there is currently no 'one perfect' solution that covers all of these autofluorescent nuisances. The autofluorescence from lipofuscin seems to need  quenching with different mechanism to formalin fixation and most other tissue elements. Popular home-brew solutions include Sudan Black, sodium borohydride, ammonia in ethanol, Toluidine blue and copper sulphate solutions. 1 These solutions can help, but also have their limitations, depending on the source of the autofluorescence. Some are not nice chemicals to work with and some have their own fluorescence under certain filter sets, limiting their application with similar fluorophores. 

Commercial preparations also tend to be specific for one of the groups of autofluorescence-  such as Vector Laboratories' TrueView is excellent for most types of autofluorescence except lipofuscin, and Biotium's TrueBlack is primarily marketed as a quencher of lipofuscin autofluorescence. It is possible however to combine some methods to achieve blocking of as much autofluorescence as possible, as in this method.

Sometimes it is not easy to determine what type of autofluorescence is being seen – or if it is a combination. Lipofuscin tends to accumulate in vesicles of damaged tissues, and those which have degenerative diseases, such as Alzheimer’s, and Parkinson’s, however these tissues may also be formalin fixed!

If the source of the autofluorescence is not determined, do contact our Technical Services for assistance.