Biotin and Digoxigenin as Labels for Light and Electron Microscopy in Situ Hybridization Probes: Where Do We Stand?

Abstract

Biotin was recently applied to detect cellular DNA or RNA. In combination with avidin, streptavidin or antibody, it can be conjugated with fluorescent dye, enzyme, ferritin, or gold. However, emphasis has recently been placed on the false-positive results that are obtained when this probe is used, because endogenous biotin may sometimes interfere with specific signals. Digoxigenin appears to be an interesting alternative because it is present exclusively in Digitalis plants as a secondary metabolite. We discuss in this review the efficiency and the respective advantages and disavantages of these two probes for in situ hybridization, mainly at the electron microscopic level.

Methods for visualizing RNA processing and transport pathways in living cells

“…Fixation and cell pretreatment procedures may disturb the localization of RNAs and to some extent deteriorate cell morphology. Furthermore, some of the antibodies that are routinely used to detect haptenized probes following hybridization are shown to (cross)react with endogenous cytoplasmic components (Chevalier et al 1997;Dirks and Raap 1998). In addition, information about the dynamics of RNA synthesis and transport can be obtained best when these processes are studied in living cells.…”

Detection of endogenous biotin in various tissues: novel functions in the hippocampus and implications for its use in avidin-biotin technology

“…However, the majority of studies employing avidin-biotin-based techniques have not been controlled for the potential interference of endogenous biotin. This problem has so far mainly been addressed in histochemical studies (including in situ hybridization) (Chevalier et al 1997). However, it has been postulated that biotin-containing proteins are likely to be destroyed by formalin-fixation or paraffin-embedding procedures, suggesting that the use of avidin-biotin technology does not produce artifactual binding to fixed tissues or cells (Hsu 1990).…”

“…However, it has been postulated that biotin-containing proteins are likely to be destroyed by formalin-fixation or paraffin-embedding procedures, suggesting that the use of avidin-biotin technology does not produce artifactual binding to fixed tissues or cells (Hsu 1990). Therefore, although scattered reports have pointed out the occurrence of interference of endogenous biotin in numerous tissues such as human salivary gland (Cauli et al 1994), fish retina (Bhattacharjee et al 1997), human ovary (Seidman et al 1995), and rat kidney (Yi et al 1995), the problem is still considered to be limited to particular cellular and subcellular sites (Chevalier et al 1997). The broad relevance of endogenous biotin in virtually all biological materials has not been adequately recognized.…”

Staining of the Midbody by an Anti-digoxin-specific Antibody

“…In this respect it is also important that there is no endogenous source of the haptens present in cells. However, endogenous biotin has been reported to be present in cells in sufficiently high levels to interfere with specific in situ hybridization signals when biotinylated probes are used (Varma et al 1994;Chevalier et al 1997). Mitochondria, in particular, proved to be rich in biotin.…”

“…Mitochondria, in particular, proved to be rich in biotin. For this reason, the use of digoxigenin-or estradiol-labeled probes has been recommended for RNA in situ hybridization studies (Chevalier et al 1997;Van de Corput et al 1997). …”