We just completed a white paper with the Benaroya Research Institute, Histology & Imaging Core Lab to help experiment development, and we're interested in hearing other researchers experiences.
http://bit.ly/IHCmultRG
Multicolor enzymatic assays for FFPE are challenging. One needs to adapt the protocols used for single stainings. DAB is for several reasons not always the most optimal chromogen for multispectral imaging. The properties of other chromogens than DAB are less well known. Chromogens like fast red appear to yield less reproducible results than DAB. We encountered in several stainings suboptimal inter-run reproducibility with fast red.
Are other chromogens more reproducible ?
you can use Vina Green instead of fast red. no suboptimal results you will obtain. What do you think about this attached image...DAB-Vina Green...
here a voice of a clinical histolab. We use Roche-Ventana instruments for IHC with polymer detection kits. There is a simple way of combining DAB and Red (company's colour). The first part of the protocol is the usual protocol with deparaffination and HIER until DAB-developement. Then there is a heating step in between. It's like a second run of HIER. This leads to the denaturation of any bound antibody, but DAB is still fine. Afterward one goes ahead with the Red-polymer detection kit based on alkaline phosphatase. Result is brown and red combination. The red colour is sensible to too long ethanol baths, so we just let the slides dry and coverslip with resin media.
Thank you all for the contributions. A lot of researchers we speak to do struggle with the stability of chromogens other than DAB, in particular red alkaline phosphatase based chromogens. Thanks for the suggestion of Vina Green, and the nice image, I would love to see some more. We do have a selection of chromogens some that are mentioned in the whitepaper. Certainly look forward to seeing more stable chromogens being developed in the future.
http://www.abcam.com/kits/chromogens-and-enhancers
Yes. There is a commercially available duel antibody p16 and Ki67 using DAB and fast red as chromogen. It seems difficult to mount these slides in DPX as fast red dissolves in alcohol on dehydration step.I would like to suggest multiplexing using quantom dots.
Liquid Permanent Red (LPR) is often used with DAB, but we have noticed that VinaGreen (HRP) and LPR (AP) make a good combination for double stainings when xylene-based mounting is used. And there is no need for HIER step in between, as the enzymes are different, meaning that you can put two antibodies at the same time, secondaries at the same time, and then do LPR first and VinaGreen second.
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