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HCR™ RNA-CISH is natively compatible with IHC

Written by
The MI Team

One of the important benefits of HCR™ RNA-CISH is the high performance achieved without the harsh protease pretreatment required by other RNA-CISH methods.

bDNA RNA-CISH typically requires a protease pretreatment step to digest proteins so that large reagents can penetrate the sample. This digestion step comes at a cost both by damaging the tissue morphology and by making protein targets challenging or impossible to image in the same sample.

Because HCR™ RNA-CISH is based on dynamic nanotechnology and uses small reagents, no protease pretreatment is needed for reagents to penetrate the sample, tissue morphology is preserved, and RNA/protein co-detection is straightforward.

Protease pretreatment damages protein targets

To illustrate how desirable it is to avoid protease pretreatment, the images below display HCR™ RNA-CISH + IHC with no protease (left) and HCR™ RNA-CISH + IHC with a protease pretreatment step borrowed from a bDNA RNA-CISH kit made by another company (right): 

In each image, the RNA target is in brown, and the protein target is in red. The protease significantly damages the protein target and dramatically reduces protein staining (right). With HCR™ RNA-CISH + IHC, protease pretreatment is unnecessary, so RNA and protein targets can be imaged with native compatibility (left).

What does HCR™ RNA-CISH's native compatibility with IHC mean? 

Proteases are enzymes that indiscriminately break apart proteins. Although all proteins are impacted, there are certain protein targets that are especially sensitive to protease digestion, including Ki67, CD79, and Bcl2. Because HCR™ RNA-CISH reagents are small and can readily diffuse into samples, the protease digestion step is omitted for most sample types. As a result, proteins are undamaged and ready for plug-and-play co-detection using your IHC method of choice.

What is the HCR™ RNA-CISH + IHC workflow? 

2-step approach: HCR™ RNA-CISH (HRP) → IHC (AP). With the 2-step approach, HRP is used for RNA imaging and AP is used for protein imaging.

3-step approach: HCR™ RNA-CISH (HRP) → HRP Blocker → IHC (HRP). With the 3-step approach, HRP is used for both RNA and protein imaging with an intermediate HRP blocker step.

What colors provide good contrast for RNA-CISH + IHC staining?

We suggest coupling a red or brown chromogen for RNA-CISH with a green chromogen for IHC. 

Do I have to do RNA-CISH first followed by IHC, or can I do the reverse?

We recommend performing RNA-CISH first followed by IHC second as this approach leads to strong signal for both target types.

Contact info@molecularinstruments.com for more information.