Frequently Asked Questions (FAQ)
/x-tad-bigger>Associate Professor of Clinical Pathology
Institute for Cancer Genetics, Columbia University
New York, USA
Which is the most sensitive method? IF or IHC?
IHC allows the greatest sensitivity because of a combination of reasons:
Excellent signal to background ratio, for high density antigens (>10K molecules/cell), with a simple indirect IHC. An equivalent IF immunostaining is less sensitive because none of the fluorochrome for which filters are commonly available (except possibly Cy-3) matches the sensitivity and the signal-to-background ratio. Fluorochromes such as Pe or Cy-5 / APC, which are used in FCM and have very high quantum yield, either fade very rapidly (Pe) or have special filter requirements, not usually found on epifluorescence microscopes.
For low density antigens (>2K,<10K molecules / cell), the only reliable and diffusely available signal amplification methods are in IHC.
NOTE: routine IHC or IF may not detect antigen below 2K molecules / cell Ref. Flow cytometry sensitivity is approximately 1K molecules / cell and up.