Authors
Anna Polesskaya
Summary
The protocol of in situ hybridization to detect miRNA in skeletal muscle was optimized in our laboratory. We are very grateful to Dr. Stefano Shiaffino, and to Ambion, for useful technical advice. The detailed version of the protocol is available on request. Briefly, the tibialis anterior (TA) muscle was frozen in Tissue-Tek OCT reagent, cryo-sections (12 µM) were prepared, de-proteinized and acetylated as described in (De Nardi et al., J. Cell. Biol., 1993). Digoxigenin (DIG) – labeled miRNA probes were prepared according to the instructions for the mirVana Probe Construction kit (Ambion). Muscle sections were incubated with specific miRNA probes (500 ng/ml) overnight at 38 oC, washed with 2XSSC at 38 oC for 20 min, and treated with 400 unit/ml RNase A at 37 oC for 30 min. After two washes with PBS, the slides were incubated with FITC-coupled anti-DIG antibody (Roche) for 4 hours at room temperature, washed, rinsed with 200 ng/ml Hoecsht 33258 in PBS, mounted on glass slides with VectaShield (BioValley), and analyzed by fluorescent microscopy.Further details
The protocol was published on Protocol Exchange in 2006. To see the entire protocol, click on the source link.Advertisement
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