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autofluorescence level by quantitative analysis of identically sized image areas that
display essentially pancreatic acini (Tab. 2). Toluidine Blue treatment is able to
quench autofluorescence emanating from basal areas of acini and loose connective
tissue, but only with application of the FITC filter set. A reduction of fluorescence by
more than 80 % in acini rich areas was measured, which is however, still less
efficient compared to the parallel performed Sudan Black approach (Tab. 2, FITC
filter set). In contrary, we observed an increase in fluorescence intensity of exocrine
tissue and connective fibers when Toluidine Blue treated samples were used with
DAPI filters, which was even more evident with Cy3 and Texas Red filter sets (Fig. 6
A-F, Texas Red and connective tissue fibers not shown). A combination of Toluidine
Blue and Sudan Black treatment did not provide any improvement to the Sudan
Black procedure alone but resulted in enhanced autofluorescence especially with
Cy3 and Texas Red filter sets (Fig. 6 G-L; Tab. 2).
Application of cupric sulphate in concentrations of 10 to 20 mM with formalin-fixed
pancreatic tissue only insufficiently suppressed autofluorescence (data not shown),
but concentrations of 50 and 100 mM diminished autofluorescence noticeable (Fig. 7
A-F; Tab. 2). The specific immunolabeling with Alexa Fluor 488-conjugated
antibodies in acinar cells is completely masked by autofluorescence without
treatment (Fig. 7 A), and is hardly detectable after background suppression by cupric
sulphate (Fig. 7 C, E). After Sudan Black treatment, the same immunofluorescence
labeling led to a clearly visible specific signal and negligible background noise with all
filter sets (Fig. 7 G, H). Combined application of Sudan Black and 50 mM cupric
sulphate did not further enhance the Sudan Black effect but rather compromised the
signal intensity of the specific immunofluorescence (Fig. 7 I, J).
The capability of photobleaching was tested by using a UV-transilluminator at 302
nm. The autofluorescence of pancreatic tissue and especially of erythrocytes was still
on a high level after 2 hour of high intensity UV irradiation (Fig. 8) and was
diminished only to a certain degree (14.4 % to 41.7 %, depending on filter setup;
Tab. 2). No effect on the fluorescent antibody labeling was observed if tissue was
irradiated before application of antibodies. Combination of UV radiation and Sudan
Black treatment did not markedly improve the effect of Sudan Black staining alone
(data not shown).
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