Practical Virology Lab. (4B) ISH (In situ Hybridization)

In situ Hybridization (ISH): a highly sensitive and specific method detects, as target nucleic acid, the viral DNA or RNA in tissue sections or cytological preparations. It is useful in detecting the viruses that are difficult to grow. Examples: detecting HCMV DNA in urine and puffy-coat specimens from neonates and bone marrow transplant recipients. Detection of different types of HPV in cancer of the cervix.

Principle of ISH Substrate Conjugate Biotin- Probe Target DNA or mRNA 4 5 Substrate Conjugate 3 Biotin- Probe 2 Target DNA or mRNA 1

In Situ Hybridization Procedure

3 1 2 Xylen Proteinase K Oven 65ºC 99% 95% 70% Tissue DW

6 4 5 Hybridization Cover slip Oven 70ºC 7 Incubator 37ºC

Conjugate Substrate Counter stain 1 5 2 Conjugate 3 Substrate Detergent wash 4 Counter stain

Detection of IFN-y mRNA using ISH technique

HPV in CA Cervix

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