Tierärztliche Hochschule Hannover - TiHo Bibliothek elib

Introduction:
The preservation of tissue samples for immunohistochemical staining in diagnostic
histopathology, in clinical routine or for scientific application, is most commonly
performed by chemical fixation with glutaraldehyde or formaldehyde using neutral
buffered formalin. An optimal fixation protocol depends on the origin and size of the
tissue sample as well as on concentration, quality, temperature and pH of the
formalin solution. A poor performance of the fixation procedure may rigorously
compromise the quality of subsequent immunostaining (Bacallao R, 2006; Werner, et
al., 2000). In particular for pancreatic specimen an optimized fixation procedure is
most critical due to the high content of proteolytic enzymes. In order to limit autolysis
pancreatic samples are required to be processed fast, very thoroughly and without
delay after the surgical removal. On the other side, overfixation due to prolonged
incubation times in the aldehyde fixative should be always avoided (Werner, et al.,
2000). The duration of the formaldehyde fixation procedure has major impact for
both, tissue preservation and unwanted aldehyde-induced autofluorescence.
Excessive aldehyde cross-links cause an enhanced level of induced
autofluorescence, mask antigenic molecules of interest, or epitopes may be
chemically modified during the fixation reaction (Hayat, 2002; Heaney SA, 2011;
Puchtler and Meloan, 1985; Romijn, et al., 1999; Weber, et al., 2010).
We observed autofluorescence as a common problem in formalin-fixed archival
pancreatic tissue which caused recurring difficulties for the application of
fluorochrome-labels in histological immunostaining. The autofluorescence relevantly
interfered with the fluorescence labeling and thus complicated the interpretation of
results. Quantitative analysis is in particular problematic if autofluorescence widely
overlaps with the spectra of specific fluorescence signals. Unfortunately, the
observed tissue autofluorescence typically exhibits a broad spectral range of
excitation and emission wavelengths (Billinton and Knight, 2001; Clancy and Cauller,
1998; Schnell, et al., 1999). Autofluorescence of tissue specimen generally originates
either from naturally fluorescent tissue molecules or from chemically modified
molecules due to the fixation and processing procedures. Origin of natural
autofluorescence can be extracellular tissue components like collagen, elastin, or
mucin. Intracellular occurring fluorophores in various cell types were shown to
localize predominantly to mitochondria and lysosomes, and involve most notably
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