Tierärztliche Hochschule Hannover - TiHo Bibliothek elib

once briefly dipped in 70% isopropyl alcohol, to avoid precipitation of Sudan Black
grains. Excessive Sudan Black was wiped off manually from the back and along the
edges of the slides with non-pilling tissues. Before mounting or specific
immunolabeling, slides were rinsed four times briefly in PBS.
All samples shown were embedded in glycerol without supplements. However, in
order to reduce photobleaching effects during prolonged light exposition, we
recommend the use of 8% (w/v) n-propyl gallate (Sigma-Aldrich, Germany), dissolved
in 120 mM sodium acetate (pH 7.2), 90% glycerol. Aliquots of this embedding
medium can be stored in the dark, at -20ºC for several months, or kept at 4ºC for not
more than one to two weeks, until usage.
Cupric Sulphate Treatment
The slides with sections were immersed in 10, 20, 50, or 100 mM of cupric sulphate
(copper II sulphate pentahydrate, Sigma-Aldrich, Germany) solved in 50 mM
ammonium acetate, pH 5.0, for 60 to 90 minutes in the dark and were washed in PBS
before mounting (Schnell, et al., 1999). Fresh cupric sulphate solution was prepared
before each experiment.
Toluidine Blue Treatment
Tissue sections were stained for 8 hours with 0.1% (w/v) Toluidine Blue O (Carl Roth,
Germany) solution which was freshly prepared in 70% isopropyl alcohol. Afterwards
slides were briefly rinsed in 70% isopropyl alcohol and washed in PBS.
Photobleaching
Irradiation of the tissue sections was performed before removal of paraffin on a 302
nm UV transilluminator (model IL350M, 180 W, Bachofer, Germany). For this
purpose, slides were positioned for 2 hours with the tissue sections upside-down
facing the transilluminator screen, without any cover and keeping a distance of 4 mm
to the screen surface to avoid excessive heating of the specimen.
Combination of Sudan Black with other Methods
For combined Sudan Black and Toluidine Blue treatment, 0.1% (w/v) Toluidine Blue
O was dissolved in a saturated Sudan Black B solution and tissue slides were
incubated for 90 minutes. Treatment with cupric sulphate and Sudan Black was
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