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!!
First time IHC antibody optimization
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.
- rkay447 likes this