Tierärztliche Hochschule Hannover - TiHo Bibliothek elib

erythrocytes (Baschong, et al., 2001; Clancy and Cauller, 1998). A combined
treatment of sodium borohydride followed by cupric sulfate was applied in a recent
study for formaldehyde-fixed cyrosections of rat brain. This way, a considerable
green autofluorescence was reduced that was apparent in only a subpopulation of
neurons and therefore provided an inherent potential of false positives in green
fluorescence reporter studies (Spitzer, et al., 2011). Copper-mediated quenching was
also used to eliminate lipofuscin autofluorescence (Kikugawa, et al., 1997; Schnell, et
al., 1999) and to reduce autofluorescence of hemosiderin-laden macrophages in
cortical tissue (Potter, et al., 2012). Two possible quenching mechanisms were
suggested, either a static quenching in which the fluorescent molecule forms a tight
complex with copper, or collisional quenching which involves a transfer of exited
electrons to the copper ions (Potter, et al., 2012; Schnell, et al., 1999).
Other techniques make use of disazo dyes like Trypan Blue (Mosiman, et al., 1997)
or Pontamine Sky Blue (Cowen, et al., 1985). These dyes stain various constituents
in tissue section and are thought to mask or absorb autofluorescence background.
However, disazo dyes are known to fluoresce in red colour and therefore may
interfere themselves with fluorophor-linked labeling procedures in the corresponding
spectral range. The lipophilic disazo dye Sudan Black B has also been used as a
successful treatment to control autofluorescence, in particular for lipofuscin-derived
fluorescence in neural tissue (Oliveira, et al., 2010; Romijn, et al., 1999; Schnell, et
al., 1999), myocardium and bone marrow or for fluorescent granules in myeloid cells
(Baschong, et al., 2001). Recently, Sudan Black B has been shown to reduce
autofluorescence also in murine renal tissue (Sun, et al., 2011). Since intracellular
lipofuscin granules and other tissue constituents are stainable by the lipophilic dye,
the quenching of autofluorescence by Sudan Black B was suggested to arise from
masking the fluorescent tissue components locally, without interacting with them at
the chemical level (Baschong, et al., 2001; Romijn, et al., 1999; Schnell, et al., 1999).
Although Sudan Black B is chemically well characterized and the excitation and
emission spectra have been thoroughly investigated (Frederiks, 1977; Pfuller, et al.,
1977), the mechanisms of interference with autofluorescence have not been entirely
solved, e.g. if Sudan Black staining may obscure excitation or rather quench the
emitted light of the autofluorescent molecules.
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