Posthybridization washes

Posthybridization entails a series of washes with preset stringency conditions to adjust the specificity or the wanted degree of homology and to wash out the unbound probe. Hence, posthybridization washes ordinarily include specified amounts of formamide and sodium chloride and sodium citrate (SSC) stock to adjust for the degree of stringency. 

Inclusion of formamide in the HM mix and posthybridization washes lowers the melting temperature of DNA. Denaturation, hybridization, and stringency washes in formamide, therefore, helps preserve the architecture of the chromosomes, by lowering the denaturation and stringency washing temperature. 

Posthybridization washes The posthybridization washes serve to remove nonspecific or low homology-bound probe. 

Equilibrate membrane after hybridization and posthybridization washes for 1 min with a filtered buffer (0.45 p.m) containing 1 M Tris-HCI (pH 7.5) and 0.15 M NaCI. 

Five steps can be distinguished in membrane hybridization (i) immobilization of target nucleic acid (ii) prehybridization to saturate the remaining binding sites which would otherwise adsorb probe non-specifically (iii) hybridization in low stringent conditions to adsorb probe as efficiently as possible (iv) posthybridization washes to define the stringency of the hybridization and thus the specificity of the reaction (v) the detection step (Fig. 8.2). In addition, the hybridized probes can sometimes be stripped from the blots to expose the targets to other probes. 

Specimens can be covered with hybridization solution and then with a coverslip and sealed with rubber cement or they can be surrounded with clear nail polish (hydrophobic) and incubated in humid chambers. Optimized conditions, especially probe concentrations and posthybridization washes, can be narrowed down using P-labeled probes in ISH. 

You may do all posthybridization washes in the bottles, but the efficiency of removing unbound probe, and thus background staining, seems to be better when the blots are washed in larger volumes. 

For in situ hybridization on plant chromosomes, high quality spreads are needed that are free of cytoplasm and cell wall debris (see Chapters 23 and 24). The methods for chromosome pretreatments, probe denatur-ation, and hybridization and posthybridization washes are described in Chapter 26. This chapter describes the probe hybridization mix for digoxigenin-labeled probes (Section 3.1.) and the methods used to detect the sites of probe hybridization (Sections 3.2. to 3.5.). 

Follow the method of in situ hybridization as described in Chapter 26 (Sections 2.1. and 3.1. Chromosome Spread Pretreatment and Sections 2.2. and 3.2. Denaturation, Hybridization, and Posthybridization Washes. The hybridization mix containing the digoxigenin-labeled probe (described in Section 2.1. of this chapter) should be substituted at step 2 and 3, Section 3.2. 

There is no need to autoclave the solutions used for posthybridization washes. 

Incubate embryos in 1 mL of hybridization solution at 60°C overnight Posthybridize wash with each of the following for 5 min at 60°C ... 

Diethylpyrocarbonate (DEPC)-treated water. DEPC (Sigma, Poole, UK) is added to double-distilled water to a concentration of 0.1% (v/v). The solutions are shaken then autoclaved. All solutions used for steps between sectioning tissue and the first posthybridization wash are prepared in DEPC water. All subsequent steps use solutions prepared in double-distilled water. 

In contrast to some other protocols, we omit an RNase step in the posthybridization washes in amphioxus, it can reduce signal without enhancing specificity. Different results may be found in other species. 

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