Endogenous biotin

Blocking step 1 to block endogenous enzyme activity or endogenous biotin, use the corresponding blocking system (see Chap. 5) and wash in PBS or TBS for 2x3 min. 

Endogenous biotin may be a cause for nonspecific background staining (see Sect. 5.4). To eliminate this unwanted staining, apply an avidin/biotin block (for instance Avidin/Biotin blocking kit from VECTASTAIN, Cat. No. SP-2001). Usually, paraffin tissue sections are free from endogenous biotin, and this step may be omitted. 

Note because of a dramatic increase in the sensitivity this method may require an additional blocking step for inactivation of endogenous biotin in some tissue specimens (see Sect. 5.4). 

Based apparently on the same principle as described above, suitable but rather expensive kits exploiting monovalent Fab fragments to block endogenous tissue immunoglobulins have also been developed commercially, such as Mouse-on-Mouse (M.O.M. ) Kits by Vector Laboratories (http //www.vectorlabs.com/). Three Vector M.O.M. kits are available. These kits use the same blocking technology and biotin-avidin detection format, but offer a choice of using either an enzyme-based or fluorescent-based visualization method. 

Kim SH, Jung KC, Shin YK, Lee KM, Park YS, et al. 2002. The enhanced reactivity of endogenous biotin-like molecules by... 

The complexities of protocols for fluorescent and chromogenic in situ hybridization necessarily entail careful attention to controls. In particular, the possibility of native enzyme activity or the presence of endogenous biotin in the experimental tissue should be considered, though this can be addressed by exposing control tissue to the detection system in the absence of probe. The relative merits of digoxigenin versus biotin, and some of the technical problems associated with each, have been previously discussed (Chevalier et al., 1997 Luo and jackson, 1999). 

Fig. 1. Labeling of degraded chromatin by the TUNEL assay. During apoptosis endogenous, endonucleases cleave chromatin in the hnker region between nucleosomes. The resulting nucleosome multimers are labeled by TdT and a dUTP analog with a detectable label (biotin, DIG, or FITC) shown as. The additional nucleotide in the reaction (here shown as dCTP) may be any dNTP and serves to extend the labeling reaction by preventing steric hindrance by two adjacent labeled dUTPs. (Abbreviations are as in text.)...

Endogenous biotin In certain tissues, most commonly kidney and liver, background staining can be high because of the presence of endogenous biotin. This can be minimized by preincubation of the sections with a dilute avidin solution, followed by incubation with a dilute biotin solution (Vector Laboratories, Burlingame, CA) before the application of the primary antibody. 

The best food sources of biotin (Fig. 8) include yeast, liver, soy products, rice, egg yolks, nuts, fish, and chocolate (179,180). Although many foods contain biotin, the levels are normally very low. Endogenous biotin in foods is usually protein bound in general there is more free biotin in plant-based foods than in animal-based products. 

Endogenous biotin in foods is predominately protein bound and is relatively stable (180). Consequently it can be extracted under fairly harsh conditions, e.g., autoclaving in 4 M sulfuric acid for 2 hours at 120°C. Enzymatic hydrolysis with papain will also release biotin from proteins (181). Potential sample-cleanup procedures include adsorption on charcoal and/or ion-exchange chromatography (182,183). 

Endogenous biotin, distributed in a wide variety of tissues, may also cause background staining with biotin-based immunohistochemical techniques. This biotin is especially abundant in liver, whereas it is poor in the central nervous system and adipose tissue. Endogenous biotin activity is more abundant in the cytoplasm and cryostat sections but is also present in sections of paraffin-embedded tissues. This problem is largely eliminated by using streptavidin-based methods or by sequential treatment of sections (prior to staining) with 0.01-0.1% avidin followed by 0.001-0.01% biotin for 10-20 min each. The biotin problem is discussed in more detail later in this chapter. 

An alternative to the avidin-biotin technology, the EnVision +System (Dako) detection method, is recommended for universal use in diagnostic and research studies. It is based on enhanced polymer methodology. In comparison with APAAP, PAP, ChcmMatc , CSA, LABC, and SABC methods, the En Vision +System yields optimal detection (Sabattini et al., 1998). Its sensitivity is at least as good as that of Strept ABC techniques, and its use completely eliminates the problem of endogenous biotin. 

Sections (5 xm thick) of formalin-fixed and paraffin-embedded tissues are placed on slides, dried overnight at 37°C, deparaffinized with xylene, and rehydrated with descending concentrations of ethanol. They are treated with 3% hydrogen peroxide in methanol for 5-10 min and then rinsed with PBS. The slides are immersed in 10 mM citrate buffer (pH 6.0), and heated for 2 min at 100% power, followed by 8 min at 80% power. After being rinsed in PBS, the sections are incubated in an appropriate primary antibody. They are rinsed in PBS and then treated for 4-8 min with egg white avidin solution (2.5 g of egg white avidin in 0.1 mM PBS) (Ventana Medical Systems). Avidin binds to endogenous biotin. 

The avidin-biotin system was developed for detecting antigens at the electron microscope level (Heitzmann and Richards, 1974). Later Heggeness and Ash (1977) proposed the use of this system for fluorescence immunohistochemistry. Guesdon et al. (1979) proposed a variety of labeled avidin-biotin methods which were further supported by Warnke and Levy (1980). The avidin-biotin methods used today are similar to the system described by Hsu et al. (1981). This system is a significant improvement over the previous immuno-histochemical techniques. The problem of endogenous biotin is discussed on page 98. 

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