Development of a sensitive and specific in situ hybridization technique for the cellular localization of antisense...

A sensitive method has been developed for the identification and assessment of phosphorothioate oligonucleotide accumulation in dosed animal tissues using an in situ hybridization approach, which is both sequence specific yet adaptable to every antisense oligonucleotide (ASO), which has been tested to date. Hybridization is accomplished using a digoxigenin-tailed oligonucleotide probe complementary to the ASO target sequence on routinely processed paraffin sections which have been pretreated with a mild target retrieval solution. The DIG-labeled probe is amplified first with an anti-DIG:FITC antibody conjugate followed by an anti:FITC Alexa 488 antibody, then visualized using FITC epifluorescence microscopy. Fluorescent labeling of ASO drug in tissue sections by this method confirms that H&E basophilia previously observed in dosed tissues represents largely intact ASO. However, the fluorescent method enables a wider assessment of tissue distribution in a variety of tissue types due to increased sensitivity and lower signal to noise than can be obtained through an examination of H&E stained tissue sections alone.