In this study, through a combination of proteinase K digestion and autoclaving in a Trilogy solution, we found a relatively simple method that demonstrates an optimal ISH signal enhancement of CDV RNA.
The formaldehyde as a 10% neutral buffered formalin is the most widely used universal fixative because it preserves a wide range of tissues and tissue components.
However, attempts to extract usable DNA from formalin-fixed tissues for molecular biological studies have been variably successful (Srinivasan et al., 2002). The formaldehyde fixative initiates DNA denaturation (interchain hydrogen bonds break and bases unstack) at the AT-rich regions of double-stranded DNA creating sites for chemical interaction. There are four interactions of formaldehyde with DNA: 1) The first is an addition reaction. Formaldehyde is added to the nucleic acid base to form a hydroxymethyl (methylol) group (-CH2 OH). 2) The second is a slower electrophilic attack of N-methylol on an amino base to form a methylene bridge between two amino groups. 3) Formaldehyde treatment can generate AP (apurinic and apyrimidinic) sites via hydrolysis of the N-glycosylic bonds, leaving free pyrimidine and purine residues. AP sites have a highly unstable cyclic carboxonium ion that hydrolyzes rapidly to yield 2-deoxy-D-ribose. 4) Formaldehyde may also cause slow hydrolysis
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pyrimidines. When compared to the DNA isolated from frozen tissues, formalin-fixed tissues exhibit a high frequency of nonreproducible sequence alteration (Srinivasan et al., 2002; Shi et al., 2001).
Previous attempts have used thermocycling (Kim and Chae, 2003), microwaving (Lan et al. 1996; Gaedke et al. 1997) or autoclaving (Relf et al. 2002) to enhance the ISH signal. Recent employment of the HIAR has been shown to enhance the extraction of nucleic acid or increase the efficiency of subsequent ISH detection of a target sequence. The process of crosslinking makes probe access to the target sequence difficult. Therefore, tissues must be digested to improve probe access to the specific mRNA while minimizing loss of mRNA and tissue morphology. Many such digestion strategies have been employed to permeabilize fixed cells or tissues using acids, detergents, alcohols, and enzymes such as proteinase K, pronase, and pepsin.
However, this step remains problematic in that each tissue type requires a different set of digestion conditions (Mcquaid et al., 1990; Lan et al. 1996; Kim and Chae, 2003;
Shi et al., 2001; Weise et al., 2005; Yamashita 2007). Heating cleaves inter- and intra-crosslinks in proteins and nucleic acids, and since a gel-like structure formed by the crosslinks is destroyed and the macromolecules are partially extracted, antibodies can easily penetrate into tissue sections and the immunoreaction is greatly intensified
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The HIAR Trilogy solution is a novel product that has been shown to have good antigen retrieval effects for the immunohistochemical labelling of RNA viruses (Faoláin et al., 2005; Ward et al., 2006; Liang et al., 2007); however, due to the advantage of combining deparaffinisation, rehydration, and the retrieval of antigens during pressure cooking, this solution has never been used in an ISH protocol. In this study, Trilogy solution was found to be the idealist for retrieval of RNA in comparison to other solutions. The fact that the HIAR effect could be used as an approach to enhance the extraction of nucleic acids or to increase the efficiency of subsequent detection of a target sequence was not emphasized until recently (Shi et al., 2001; Kim and Chae, 2003). Trilogy solution is a novel product that combines deparaffinization, rehydration, and unmasking of antigens during pressure cooking (Faoláin et al., 2005). In recent reports, Trilogy solution has been shown to have good antigen retrieval effects for the immunohistochemical labelling of RNA viruses (Ward et al., 2006; Liang et al., 2007) or cancer diagnosis (Kuo et al., 2006) but never been tried in ISH protocol, which has lead to the development of novel ISH labelling protocols that are especially important for retrospective studies. The HIAR effect (Mcquaid et al., 1990; Kim and Chae, 2003) can greatly enhance the ISH signal and provides a simple detection method in formalin-fixed, paraffin -embedded tissues.
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consideration. The pH valve of the antigen retrieval solution is another important co-factor (Shi et al., 2001; Ramos-Vara 2005). Some antigens will be retrieved only with high pH solution, others with a wide range of pH. Most antigens, HIAR with 0.01M sodium citrate buffer (pH 6.0) will give satisfactory results and good cell morphology (Ramos-Vara 2005). However, the pH values of the Trilogy (7.69), TBS (8.22), H3301 (9.14), and Dako S1700 retrieval working solutions (9.22) were from neutral to weak basic pH valve in the present study. S1700 and H3301 retrieval solution had very similar pH value but gave totally different results. The citrate-based Vector H3301 retrieval solution combined with autoclave pre-treatment showed the most severe destruction of the tissue morphology.
The results demonstrated that a simple and modified combined pre-treatment of HIAR autoclaving the tissue sections in antigen retrieval buffer and proteinase K digestion resulted in stronger hybridization signals than proteinase K digestion alone.
Importantly, autoclaving in either Trilogy solution or TBS resulted in strong hybridization signals of major organs, including spleen, urinary bladder, and lung, infected by CDV with mild to no tissue morphological damage. Similar retrieval effects of autoclaving in Trilogy solution for ISH were also noted in cerebellar sections (data not shown). Since formalin is the most commonly used fixative in
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pathogenesis study of CDV infection. However, most RNA viruses are low-copy infections; and thus, RT in situ PCR is often the best method available to detect the virus in situ (Nuovo, 1995) and will be tried in the future to compare the results in this study.