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First time IHC antibody optimization


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#1 rkay447

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Posted 08 May 2018 - 12:31 PM

I am taking a stab at IHC for the first time and need to optimize two antibodies on two different xenograph tissues.  I have optimized these two antibodies for IF on cultured cells and was just wondering if there's a general rule of thumb on what dilutions to expect to work in IHC.  Or should the dilutions that worked in IF be appropriate for IHC?  Thank you!!



#2 bob1

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Posted 09 May 2018 - 12:01 PM

It's been a while since I did this sort of thing, but I think I recall that, dependent on the fixative and fix duration, IHC will generally need lower dilutions (e.g. if you use 1:1000 for IF, then plan on 1:500 or 1:250 for IHC) due to high levels of cross-linking. The sections are also thicker in general, so the incubation times need to be a bit longer to allow full penetration.






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