Wetzel, Kristie and Capodieci, Paola (2016) Dual ISH-IHC. Publication on website acdbio.com/dual-ISH-IHC.

Abstract

Interview with Kristie Wetzel
Can you tell us about your work and why you developed a dual protocol?
I work as an investigator in the developmental and molecular pathways lab at Novartis. Our group provides immunohistochemistry (IHC) and in situ hybridization (ISH) services not just to our own lab but also to other groups working in diverse disease areas. This means that we get many different sample types depending on the focus of the particular study; it might be tissue from human, mouse, rat...
We developed a dual ISH-IHC protocol because fundamentally, we want to be able to see where the mRNA is accumulating. We want to identify which tissue and which cell type is producing it. That’s why RNAscope® ISH is really important in our work and that’s why we are using it. There are also certain scenarios where our targets don’t have very good antibodies, or using antibodies is difficult such as when profiling a secreted protein. What we get when we combine ISH and IHC is the ability to identify both the particular cell type by IHC and the mRNA profile by ISH. This information is of great interest to us.
How was your dual ISH-IHC protocol set up? Were there any challenges?
We tested a protocol where we first perfom the chromogenic RNAscope® assay (RNAscope®VS Red) followed by immunofluorescence staining. Our target was a lysozyme protein. Both of the reactions were automated on the Ventana Discovery system. To set this up, we first confirmed that the RNAscope® assay was working on our tissue samples and then we optimized our IHC protocols. We decided to do ISH followed by IHC because it was important to digest the tissue for our lysozyme antibody and the RNAscope® protocol has an antigen retrieval step followed by a protease treatment step. It took us a number of weeks and a number of iterations before we really got the protocol optimized but once we did, the resulting staining was really beautiful and provide a interesting data combination to see.
Our major challenge was optimizing the IHC protocol as protein stability is affected by the RNAscope® pretreatments. It was important to make sure that the IHC was optimized independently of the RNAscope® ISH first before combining it in the dual reaction.
Do you have any recommendations you’d like to share?
I definitely recommend working out the individual protocols separately before combining them. You have to have a good signal individually first, but you also need to realize that even if they’re both working well individually, you’re still going to need to tweak and optimize once you combine them. The IHC protocol is definitely going to need optimizing due to the RNAscope® pretreatment - it’s also important to determine the optimal RNAscope® pretreatment protocol for each tissue type you have. Once this is established, it’s not going to change.
Another thing I would recommend is following the RNAscope® protocol to the letter! It works very well if you do this.

Figure 2. Dual ISH-IHC staining using chromogen labeled enzymes to identify mRNA expression in the
stem cells, and protein expression in the Paneth cells in the mouse gut. Provided by Kristie
Wetzel.

Item Type:	Article
Date Deposited:	10 May 2016 23:45
Last Modified:	10 May 2016 23:45
URI:	https://oak.novartis.com/id/eprint/27971