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

2. 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.

3. Principle of ISH Substrate Conjugate Biotin- Probe Target DNA or mRNA4 5
Substrate
Conjugate 3
Biotin-
Probe 2
Target
DNA or
mRNA 1

4. In Situ Hybridization Procedure

5. 3 1 2
Xylen
Proteinase K
Oven
65ºC
99%
95%
70%
Tissue DW

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

7. Conjugate Substrate Counter stain1 5 2
Conjugate 3
Substrate
Detergent
wash 4
Counter stain

8. Detection of IFN-y mRNA using ISH technique

9. HPV in CA Cervix

10. THANK YOU