Tierärztliche Hochschule Hannover - TiHo Bibliothek elib

Results:
Evaluation of intrinsic fluorescence of formalin fixed pancreatic tissue
We analyzed the autofluorescence of formalin-fixed, paraffin-embedded human
pancreatic tissue sections using reflected light fluorescence microscopy. To quantify
the intensity of autofluorescence in the critical range of wavelengths we applied
several epifluorescence filter sets which are frequently used for the detection of
common fluorophores in microscopy (for specification of filter sets see Tab. 1).
We first determined the overall distribution of autofluorescence in formalin-fixed
pancreatic tissue and, in more detail, its subcellular localization within pancreatic
acini. Using filter sets suitable for the detection of the fluorophores DAPI, FITC, Cy3
(Fig. 1 A-C) or Texas Red (data not shown), respectively, we observed a nonhomogenous
distribution of autofluorescence within tissue sections. The spatial
distribution and resulting pattern of autofluorescence was nearly identical with the
different excitation and emission spectra applied, however, the intensity of signals
varies. With each of the filter sets, we detected relatively bright fluorescence
originating particularly from nuclei and the zymogene granule-rich apical areas of
acinus cells (Fig. 1). Quantitative analysis of the signal intensity was performed by
defining a narrow region of interest (Fig. 1 D, marked area between white lines) and
calculating the mean intensity profile along this region (Fig. 1 E). By this means,
nuclei could be clearly identified as peaks within the intensity profile. The acinus
lumen was almost free of detectable autofluorescence while the granule-charged
cytoplasm of acinar cells exhibited signal plateaus with superimposed flickering
amplitude. Overall, autofluorescence emitted from pancreatic acini was of
approximately 3 to 4 times higher intensity than autofluorescence detected in loose
connective tissue (e.g. of a pancreatic septum, data not shown).
Reduction of autofluorescence using Sudan Black treatment
For immunofluorescence microscopic studies of formalin-fixed tissue sections an
adequate background reduction is required in order to avoid erroneous results and to
improve the signal-to-noise ratio. An optimized procedure to stain pancreatic tissue
samples with Sudan Black B was established to efficiently quench autofluorescence
of human pancreatic tissue sections. The intensity of tissue staining by Sudan Black
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