Dr. Giorgio Cattoretti
Associate Professor of Clinical Pathology
Institute for Cancer Genetics, Columbia University
New York, USA

Which is the most sensitive method? IF or IHC?

Answer:

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.