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10.1 What types of molecule can be demonstrated using ISH and to what type of preparations can the method be applied?
Answer: DNA and RNA (principally mRNA and microRNA) sequences can be demonstrated using ISH. The method can be applied to routinely prepared cytology preparations and formalin fixed paraffin embedded tissue sections. It is also applied to cell culture and whole mount preparations for research.
10.2 What are the three types of nucleic acid backbones and hapten labels that can be employed for ISH?
Answer: With respect to backbones these are; 1) the standard sugar/phosphate of DNA or RNA, 2) modification of the ribose ring of DNA to make a locked nucleic acid and 3) the substitution of a peptide moiety for the phosphates of DNA to give a peptide nucleic acid probe. Biotin, digoxigenin and fluorescein derivatives are the most commonly used hapten labels.
10.3 What are the key criteria for optimal cell and tissue preparation of samples for ISH?
Answer: The key criteria are; 1) fixation to conserve cytology/morphology of the cell/tissue preparations and 2) pre-treatment to expose masked nucleic acid target sequences after the use of formalin containing fixatives. Too short fixation may not retain target sequence, whilst too long fixation can irretrievably degrade or mask the target. Pre-treatment must be carefully controlled. Under exposure of the target would give a negative or visualisation; over exposure could liberate the target sequence or result in compromised cytology/morphology.
10.4 How do probe concentration, temperature of hybridization, monovalent cation concentration and formamide interact to affect the specificity of hybrid formation?
Answer: Too low probe concentration can result non-saturation of the target, too high concentration may lead to off target hybridization to nucleic acid sequences that share partial sequence homology. Increase in temperature and low monovalent cation concentration promotes high specificity of hybrid formation. Conversely, lowering hybridization temperature and increasing monovalent cation concentration will increase the rate of hybrid formation, but could allow for partial homologous sequence hybrid formation. As a helix destabilising reagent, formamide allows hybridization temperature to be lowered to levels that promote the conservation of the cytology/morphology of samples. When using non-commercial probes all of these factors must be experimentally determined to ensure high specificity of hybrid formation.
10.5 What chromogenic end points options are available for the completion of an ISH technique?
Answer: Several end points are available. These include the demonstration peroxidase and alkaline phosphatase enzyme activity (CISH) and the deposition of metallic silver (SISH). For the former both alcohol resistant (peroxidase with diaminobenzidene) and alcohol soluble precipitates (formazan of the NBT/BCIP reaction with alkaline phosphatase) are formed. These coloured precipitates contrast with black silver deposits of SISH and can form the basis of sequential or simultaneous demonstration of two nucleic acid targets in the same preparation.
10.6 What type of sample preparation could be used as a positive control for assessment of runs?
Answer: The preparation could be a section of a multi-block of cell lines containing different copy number of the target sequence. Alternatively, it could be a tissue section that contains cells with a moderate expression level of the target. The use of preparations that contain very little or an abundance of the nucleic acid target are not suitable as they will fail to detect assay drift.
10.7 What are the chief distinguishing features of TSA and branched DNA methods in comparison with standard CISH as illustrated in Box 10.1?
Answer: TSA and branched DNA methods offer a significant amplification of target sequence detection in comparison with the use of standard CISH methods. TSA amplification is based on the horse radish peroxidase catalysed deposition of an activated hapten tyramide derivative. The hapten is then demonstrated. Branched DNA also provides for significant amplification. This is achieved through initial hybridization of multiple ‘Z’ probe couplets to the target sequence, conferring a specificity advantage. This is followed by repeated hybridizations that eventually result in the incorporation of many more enzyme molecules than are available for cycling of a substrate than with standard CISH detection systems.