I have to perform double staining by coupling ISH for EBER (non-coding RNA of EBV) and a brightfield IHC for a protein potentially expressed by infected cells. I work on sections of autopsy brain tissue.
By sequencing ISH and then IHC, like the protocol I've been following for some time, the signal for the protein disappears. I therefore think that antigenicity is not maintained after the ISH procedure. So I thought about doing IHC first and then ISH for EBER, hoping to keep the IHC signal, but I can't find a protocol that follows this sequence, first IHC for a protein and then ISH for a transcript.
Do any of you have any suggestions?
Thank you
Barbara Serafini You can go through this technical sheet using RNAscope.
https://resources.bio-techne.com/bio-techne-assets/images/resources/ish-ihc-app-note-wr.pdf
Thank you Justin, I know this technique. The steps are: "There are three general guidelines when performing the dual ISH-IHC/ IF workflow. First, it is highly recommended that the RNAscope ISH assay be performed before the IHC/IF assay" . Etc etc.
So, this is not what I'm searching for.
thank you all the same
Thank you Narendran, this is the classic protocl to perform double staining combining ISH and IHC. My problem is that the protein that I need to visualize in IHC is destroyed by the ISH procedure, so when a I perform the double staing I only can see the ISH signal. From this my question about the possibility to perform first IHC and then ISH. But so I think that my question was not enterely clear.
Thak you for your time.
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