General Procedures For

3.1. SPECIMEN FIXATION With regard to in situ molecular detection techniques, the most basic influencing factor is specimen fixation, which serves to preserve morphology and retain the necessary target nucleotide sequences. The common fixatives in use are formalin, Carnoy's fixative (methanol : acetic acid), Bouin's solution (picric acid), and ethanol. Of these, buffered formalin seems to best preserve DNA and mRNA in a tissue section (21). Formalin brings about target protein and nucleic acid fixation via crosslinking the protein amino groups and hydroxymethylation of nucleic acids (22). The efficiency of formalin to preserve especially the low abundance RNA species in a tissue could be further enhanced by adjusting the pH of formalin to alkaline conditions (23). Buoin's solution is generally not recommended for ISH/FISH applications. For cytological preparations on the other hand, simple air-drying or gentle fixation with methanol or Carnoy's fixative appear to work comparably (24). The fact emphasized by different studies comparing a variety of fixation methods is that an immediate fixation of a specimen preserves the target with utmost efficacy.

3.2. SPECIMEN PRETREATMENT Most laboratory cell preparations such as cultured cell lines or blood lymphocytes usually do not have contaminating debris or cytoplasm from lysed cells or extracellular matrix (ECM) components and so forth and, hence might not need any pretreatment before hybridization with the probe. However, most types of target cell in a clinical specimen such as amniocytes, which have a layer of contaminating proteins, might need a pretreatment for optimal hybridization results. Also, the tissues fixed with crosslink-ing agents like formalin need pretreatment to unmask the target nucleotide sequences. Usually, the pretreatment protocol involves one or more of the following steps: (1) permeabaliza-tion with isothiocyanate solution or mild periodic acid solution, (2) mild acid hydrolysis with 0.05 M to 0.1 M HCl, and (3) pro-teolytic enzyme treatment primarily with pepsin, trypsin, or proteinase K (20). Previous studies have noted that the protease treatment for 20-30 min offers definite signal enhancement. However, an, overextended protease treatment could compromise cellular morphology (25). Additionally, a pretreatment with two to three short cycles (3-5 min each) of microwave heating in citrate buffer (pH 6.0) has been shown to enhance the hybridization outcome (25,26).

Clearly, several options are available for pretreatment. The combination of different options would need to be optimized in each individual laboratory, as the initial specimen processing conditions vary greatly from place to place. When the specimen is pretreated, a mild postfixation with low-concentration paraformaldehyde is highly desirable to prevent the excessive loss of a target sequence and/or occasionally of the specimen itself (20). With cell preparations, additional dehydration through graded alcohol is beneficial.

3.3. HYBRIDIZATION AND VISUALIZATION A variety of probe designs have been successfully used in ISH as reviewed earlier by McNicol (20). Briefly, the double-stranded DNA probes are generated by cloning and amplification. These large double-stranded probes require denaturation to obtain a single-stranded DNA. On the other hand, a single-stranded complementary DNA (cDNA) probe could be generated directly by reverse transcribing mRNA with a specific primer followed by amplification employing polymerase chain reaction (PCR). PCR amplification of a DNA template using antisense primer provides another option. The advantage of the cDNA probe lies in its ability to directly hybridize without prior denaturation. An example of a PCR-generated single-stranded cDNA probe is shown in Fig. 1a. Alternatively, antisenseRNA or synthetic oligonucleotide sequences could also be used as probes. Some studies indicate that the antisenseRNA probes might offer higher sensitivity than the single-stranded cDNA probes in detecting low-abundance mRNA species (27,28). However this proposition should be validated for individual situations.

Regardless of the probe type, they could be directly labeled using nucleotides containing radioisotope as originally described or conjugated with fluorescent chemical labels. Such labels could either be incorporated during the probe synthesis or 3'-end labeled with terminal transferase following their synthesis. The most common radioisotopes used are 32P, 33P, 3H, and 35S. Barring the safety concerns with their use, these labels do score merits for the ease in labeling and sensitivity of target detection. The use of nonisotopic fluorescent tags such as rhodamine, fluorescein, and so forth overcome the safety concerns regarding radiotisotopic labels and, in fact, according to some studies might even match comparably in sensitivity (29). Alternatively, the probes could be labeled at the 5' end with

Fig. 1. In situ hybridization for detection of mRNA. (A) Cytomegalovirus (CMV)-infected human foreskin fibroblasts expressing mRNA (brown staining) for viral immediate early protein (IEP). A PCR-amplified single-stranded biotinylated cDNA probe prepared in the author's (SM) laboratory at Rush University, Chicago, IL, was employed with avidin-biotin-horse radish peroxide and diaminobenzidine as a detection system. Original magnification: x1000. (B) A paraffin-embedded breast cancer tissue section shows the presence of Histone-H3 mRNA (dark blue staining) (Courtesy; of Dr. Smitha Sivaraman, Rush University and Dr. Charuhas Deshpande and Dr. Sunil Badve of Northwestern University, Chicago) A synthetic commercial oligonucleotide probe tagged with fluoresceinisothiocyanate (FITC) was used, which was visualized by anti-FITC antibody conjugated to alkaline phosphatase and nitroso-blue substrate. Original magnification: X1000. (Figure appears in color in insert following p. 172.)

Fig. 1. In situ hybridization for detection of mRNA. (A) Cytomegalovirus (CMV)-infected human foreskin fibroblasts expressing mRNA (brown staining) for viral immediate early protein (IEP). A PCR-amplified single-stranded biotinylated cDNA probe prepared in the author's (SM) laboratory at Rush University, Chicago, IL, was employed with avidin-biotin-horse radish peroxide and diaminobenzidine as a detection system. Original magnification: x1000. (B) A paraffin-embedded breast cancer tissue section shows the presence of Histone-H3 mRNA (dark blue staining) (Courtesy; of Dr. Smitha Sivaraman, Rush University and Dr. Charuhas Deshpande and Dr. Sunil Badve of Northwestern University, Chicago) A synthetic commercial oligonucleotide probe tagged with fluoresceinisothiocyanate (FITC) was used, which was visualized by anti-FITC antibody conjugated to alkaline phosphatase and nitroso-blue substrate. Original magnification: X1000. (Figure appears in color in insert following p. 172.)

protein haptens such as biotin and digoxigenin (indirect probe labeling). These reporter molecules could then be detected using fluorescent- or enzyme-labeled streptavidin in the case of biotin or a specific antibody in the case of digoxigenin. Figure 1a,b illustrates the use of ISH indirect reporting systems. The indirect probe labeling approach could at times compromise the sensitivity of target detection, and the use of blocking agents like bovine serum albumin (BSA) is required to block the nonspecific binding of haptens to cellular proteins (30).

Generally, the factor that tremendously influences the specificity of probe binding is the hybridization condition of an assay (31). The hybridization stringency is, in turn, determined by several factors like pH, salt and formamide concentration, temperature, and so forth. By varying annealing temperature and/or formamide concentration in the hybridization buffer, the nonspecific cross-hybridization could easily be prevented. Use of dextran sulfate is advocated for enhancing the efficiency of specific hybridization (31). Usually, cRNA (antisenseRNA) probes appear to need longer hybridization times than cDNA probes. Competitive hybridization with an excess of unlabeled probe, probe preabsorption, use of sense-RNA probe, and RNAse or nuclease-treated specimens are examples of recommended controls for ISH (20,31).

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