Tierärztliche Hochschule Hannover - TiHo Bibliothek elib

Abstract:
High autofluorescence levels in tissue samples can entirely mask specific labeling
with fluorophores and thus compromise the validity of immunofluorescence
histochemistry. In formalin-fixed archival pancreatic tissue samples we observed
autofluorescence as a common problem mostly induced by a fixation and processing
procedure. Using epifluorescence microscopy we analyzed the intensity as well as
the spatial distribution of autofluorescence in human pancreatic tissue and
demonstrated an efficient method to reduce the unwanted light emission. The
optimized protocol which uses the dye Sudan Black B is demonstrated to stain
particularly those pancreatic tissue areas which exhibit notably bright
autofluorescence. As a result, tissue autofluorescence is reduced to a low and
intensity-equalized background level. Implementing quantitative image analysis and
using a range of different microscopic fluorescence filter setups autofluorescence is
shown to be suppressed by 65 % to more than 90 %. The application of our
procedure did not affect specific immunofluorescence labeling nor tissue integrity. As
a clear result of Sudan Black treatment, a tremendous improvement of the signal-tonoise
ratio was achieved, which is a basic prerequisite for detection, visualization and
quantitation of specific fluorescent labels. Other methods applied such as tissue
treatment with cupric sulphate, Toluidine Blue and UV-irradiation, or combinations of
these with Sudan Black B, were not able to match or surpass the superiority of the
Sudan Black B staining approach. This easy to perform method allows a well defined
and reliable fluorescence labeling in pancreatic tissue sections. Due to its capability
to drastically reduce autofluorescence of pancreatic specimen this technique can
clearly improve qualitative as well as quantitative analysis of immunohistochemical
stainings and rescue overfixated tissues for immunofluorescence application.
Abbreviations:
BSA bovine serum albumin, CAPS N-cyclohexyl-3-aminopropanesulfonic acid, CCD
cooled coupled device, Cy3 cyanine3, DABCO 1,4-diazobicyclo 2,2,2 octane, DAPI
4',6-diamidino-2-phenylindole, EDTA ethylenediaminetetraacetic acid, FITC
fluoresceinisothiocyanine, IgG immunoglobuline G, min minute, mM millimolar,
MOPS 3-(N-morpholino)propanesulfonic acid, NADH nicotinamide adenine
dinucleotide, PBS phosphate buffered saline, SB Sudan Black B, Tris
tris(hydroxymethyl)aminomethane
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