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was visualized using bright field microscopy (Fig. 2 A-G). In particular pancreatic
acini exhibited a good optical contrast due to a selective grey-to-black staining.
Acinar cell nuclei appeared rather moderately stained and were surrounded by an
intensively pigmented cytoplasm which is enriched in zymogene granules. The
overall staining intensity clearly increased with prolonged incubation times of the
tissue samples in Sudan Black solution, reaching a maximum after about 90 to 120
minutes (Fig. 2 F, G). The resulting intensity of autofluorescence of these Sudan
Black treated specimen was evaluated with all four fluorescence filter sets. We
exemplary illustrat data using the DAPI filter set (Fig. 2 H-P), albeit the other filter
sets showed similar characteristics. Incubation times of 15 and 30 minutes led to a
clearly visible reduction of autofluorescence (Fig. 2 I, J). However, a further
improvement could be achieved with prolonged incubation in Sudan Black solution
(Fig. 2 K-N). As demonstrated by quantitative image analysis, autofluorescence
intensity decreases remarkably with enhanced time of tissue incubation in Sudan
Black B (Fig. 2 P). Within the indicated tissue region displaying primarily pancreatic
acini, an overall background reduction of about 59% was achieved after 15 to 30
minutes and 60 to 90 minutes of Sudan Black treatment resulted in approximately
80% reduction of total autofluorescence (Fig. 2, DAPI filter setup). Even longer
treatments hardly led to further improvements. We decided to use the incubation time
of 90 minutes for our standard protocol in order to assure maximal efficiency of
Sudan Black treatment. It is also worth noting that the autofluorescence which
emitted from cell nuclei seems to be somewhat less efficiently quenched, most likely
due to the lower staining intensity of nuclei with the Sudan Black dye. As a result,
nuclei are faintly visible in fluorescence images using each of the fluorescence filter
sets (Fig. 2 K-N).
Using the optimized Sudan Black staining procedure, different emission and
excitation wavelengths were applied for a detailed quantitative analysis of the
quenching effect on autofluorescence. For this purpose we selected a pancreatic
tissue area showing a small artery as a prominent mark. To identify corresponding
areas of interest we used neighboring specimen of serial sections. Without Sudan
Black treatment, an intensive autofluorescence was observed in particular with DAPI
and Texas Red filter sets. A somewhat lower but still prominent intensity of
autofluorescence was apparent when the FITC and Cy3 Filter sets were applied (Fig.
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