Tierärztliche Hochschule Hannover - TiHo Bibliothek elib

presence of hemoglobin (Casella, et al., 2004). The excellent outcome of Sudan
Black treatment was determined quantitatively and documented by fluorescence
intensity profiles of pancreatic specimen (Fig. 1 E; 3 I-L). The peaks of
autofluorescence intensity were eliminated and autofluorescence was suppressed to
a low and outstanding homogeneous background level. In our experiments Sudan
Black treatment could reduce the intensity of the major autofluorescence peaks
between 73% (for artery wall, using Cy3 filter set) to more than 99% (for erythrocytes,
using FITC filter set) (Fig. 1E; 3 I-L). A further advantage of the Sudan Black staining
procedure is a tissue appearing rich in optical contrast. This allows easy navigation
and focussing within tissue samples using bright field microscopy which also
facilitates the observation of distinct tissue areas in fluorescence microscopy. As a
rather minor drawback Sudan Black treatment was not able to completely eliminate
the autofluorescence of acinar cell nuclei, most likely since the staining of nuclei was
not intensive enough (Fig. 2).
Other techniques used in this study to reduce autofluorescence background were
found to perform in pancreatic tissue insufficiently or interfere with specific
fluorescence labeling. Cupric sulphate was successfully used to quench lipofuscinlike
autofluorescence in neural tissue (Schnell, et al., 1999) but was not beneficial
when applied with pancreatic tissue in our study. Concentrations of more than 50 mM
cupric sulfate partially suppressed unwanted autofluorescence, but considerable less
efficiently compared to Sudan Black treatment (Tab. 2). However, in line with Schnell
et al. (1999), we observed that applications of 50 mM cupric sulphate or higher have
clearly negative impact on specific immunofluorescence signals. Although not
included in this study, we analyzed the application of sodium borohydride for
formaldehyde-fixed pancreatic tissue during previous studies (Meister, et al., 2010;
Schnekenburger, et al., 2009). This treatment was found to be not beneficial primarily
due to adverse effects on immunostaining. The dye Toluidine Blue has been widely
used as counterstain in combination with immunohistochemistry labeling
(Chelvanayagam and Beazley, 1997). Indeed, Toluidine Blue treatment was helpful
to suppress autofluorescence of acinar cells, connective tissue, and elastic fibers of
blood vessel walls, if observations were limited to the FITC filter set. Still,
autofluorescence reduction was less effective compared to the Sudan Black protocol,
which was particular evident in zymogene granule rich tissue areas (Tab. 2).
- 20 -