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effects on the fluorescent staining. When using a pretreatment with Sudan Black in
neural tissue Schnell et al. (1999) observed an even unacceptable reduction of
immunofluorescence signals. However, in our assays, in fact no obvious interference
with the performance of immunolabeling was observed if Sudan Black was applied
prior to the fluorescence staining procedure. We proved that this optimized Sudan
Black method is compatible with the immunodetection of various epitopes localized in
exocrine and endocrine pancreas and with different fluorochromes that are widely
used with immunofluorescence techniques (Fig. 5). Particular care should be taken to
select only non-interfering embedding media for the mounting of Sudan Black stained
specimen. This finding is consistent with results of Romijn et al., 1999, who further
recommended the omission of antifading agents. Importantly, we suggest in addition
to plain glycerol a newly formulated embedding medium including the antioxidant n-
propyl gallate with beneficial characteristics for the Sudan Black application.
The superiority of Sudan Black treatment for pancreatic tissue seems to arise from
the fact that the dye binds most notably to selected tissue constituents that exhibit
outstanding high autofluorescence, and thus locally reduces the emission peaks.
Sudan Black B stains a wide range of subcellular molecular structures, above all
lipids and phospholipids, but also some proteins and mucopolysaccharides. The
chemical interactions between the dye and tissue components can be manifold and
have been discussed controversial (Frederiks, 1977; Lillie and Burtner, 1953; Pfuller,
et al., 1977; Schott and Schoner, 1965). In pancreatic tissue, we identified nuclei and
zymogene granules of acinar cells to exhibit notably bright autofluorescence, besides
more ubiquitous tissue components like connective fibers and erythrocytes (Fig. 1, 3).
Clearly, Sudan Black effectively quenched autofluorescence of acinar cells which
was likely mediated by the illustrated ability to intensively stain zymogen granules
(Fig. 2), thereby obscuring the autofluorescent components. Its property to stain
intracellular granules is long known for leukocytes, dermal mast cells and also for
pancreatic B-cells (Lillie and Burtner, 1953; Rode, 1962; Sheehan and Storey, 1947;
Williams, et al., 1959). In line these observations, more recent studies confirmed that
Sudan Black is also able to mask autofluorescence of myeloid granules (Baschong,
et al., 2001). In pancreatic tissue we found that Sudan Black further stains connective
tissue fibers and erythrocytes intensively (Fig. 3 H) which clearly supports the
quenching of tissue autofluorescence and the intense fluorescence caused by the
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