Simultaneous detection of RNA and protein by in situ hybridization and immunological staining

Proteinase K is widely used in methods for detection of transcripts in biol ogical specimens by in situ hybridization (ISH). However, treatment with pr oteinase K hampers detection of RNA and protein simultaneously. We have dev eloped a method for double staining of transcripts and proteins by ISH and IHC staining in imaginal discs and embryos of Drosophila. Instead of treatm ent with proteinase K, samples are treated with ethanol plus xylene and wit h acetone. Acetone renders cell membranes permeable to probes and antibodie s without damaging tissue integrity, whereas treatment with proteinase K so metimes damages tissues. Treatment of samples with acetone allows hybridiza tion of probe with transcripts in tissue. It is also effective for immunolo gical staining of samples after ISH with a riboprobe. Thus, our method allo ws detection not only of transcripts but also of specific proteins in relat ively intact single samples.