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3 A-D). A quantitative analysis of the emitted light intensity is shown in form of
histograms (Fig. 3 I-L colored lines). Blood vessel walls and pancreatic duct walls
with the enclosing dense connective tissue rich in fibers were found to exhibit
fluorescence intensities comparable to pancreatic acini (Fig. 3 J and data not shown).
The highest degree of autofluorescence originated from erythrocytes in the arterial
lumen, which was obvious for each of the indicated filter sets (Fig. 3 A-D) resulting in
distinctive peaks of intensity in the corresponding histograms (Fig. 3 I-L). Using the
Texas Red filter set, red blood cells developed the brightest fluorescence signals
(Fig. 2 K), the intensity was twice as high as emission from arterial walls. Microscopic
observation with FITC or Cy3 filter set exhibited a similar pattern of autofluorescence
but a somewhat lower signal amplitude (Fig. 3 J, L). Using the DAPI filter setup, the
emitted light intensity from elastic fiber rich areas of vessel walls almost equals the
autofluorescence level of erythrocytes (Fig. 3 I).
After treatment with Sudan Black the tissue autofluorescence is rigorously reduced
with all tested filter sets (Fig. 3 E-G; and Fig. 3 I-L black lines of histogram). An
intensive staining with Sudan Black dye was particularly evident for erythrocytes and
elastic fibers (Fig. 3 H). In line with this observation, autofluorescence of erythrocytes
was quenched efficiently with each of the filter sets. Quantitative analysis (Fig. 3 I-L)
revealed that the peak of fluorescence intensity originating from erythrocytes was
reduced by 81% when observed with the DAPI filter setup, 92% with Cy3, more than
99% with FITC, and 97% with Texas Red filter sets. Autofluorescence intensity of the
artery wall (Fig. 3 I-L) was reduced by 73% with Cy3 to a maximum of 99% with the
FITC filter setup. Thus, as a result of Sudan Black treatment, the remaining emitted
light had a surprising low and equal intensity level throughout the imaged tissue area.
To verify the suitability of Sudan Black application for the combination with
fluorescence immunolabeling is of utmost importance. In untreated specimen
autofluorescence almost completely mask the specific fluorophor labeling of acinar
cell granules (Fig. 4 A). Application of Sudan Black treatment after the procedure for
immunolabeling clearly facilitates the visualization of specific immunofluorescence
signals, however, the signal intensity was rather moderate (Fig. 4 B). Although
quenching of the autofluorescence was similar effective, a maximal fluorescence
intensity of specific labels was preserved when Sudan Black staining was performed
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