A simple and sensitive antigen retrieval method for free-floating and slide-mounted tissue sections

J Neurosci Methods. 1999 Nov 15;93(2):149-62. doi: 10.1016/s0165-0270(99)00142-9.


The masking of antigens by aldehyde-containing fixatives or by paraffin embedding procedures is a problem for immunohistochemical studies. Enzymatic digestion, formic acid treatment, microwave heating and autoclave heating have been used to deal with this problem, with microwave heating-based antigen retrieval having become widely used as the method of choice. Microwave heating, however, has the shortcoming that it is difficult to precisely control the heating temperature and it is difficult to apply this method of heating to free-floating sections without damaging the sections. We describe here a simple, reliable and sensitive antigen retrieval method that uses water-bath heating. By this method, the temperature can be precisely controlled to yield effective antigen retrieval with minimal tissue damage in free-floating or paraffin-embedded slide-mounted sections. We found that the best results were obtained with a 30 min incubation in a 10-50 mM sodium citrate solution (pH 8.5-9.0) preheated to and maintained at 80 degrees C in a water-bath, followed by 30 min incubation in 0.3-3% nonfat dry milk to reduce nonspecfic staining. This method is highly effective for both 40 microm free floating sections, slide-mounted cryostat sections and paraffin-embedded slide-mounted sections, and it works well for tissue from diverse species (human, rat, mouse, pigeon, and zebra finch) and for diverse antigens (e.g. enkephalin, substance P, huntingtin, GluR1, GFAP, and ubiquitin). This method was also found to enhance immunolabeling in glutaraldehyde-fixed tissue that had been prepared for ultrastructural examination, without having a deleterious effect on the ultrastructure.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antigens / isolation & purification*
  • Brain / immunology
  • Brain / ultrastructure
  • Citrates / chemistry
  • Columbidae
  • Eye / immunology
  • Eye / ultrastructure
  • Histological Techniques*
  • Hot Temperature
  • Humans
  • Hydrogen-Ion Concentration
  • Immunohistochemistry / methods*
  • Mice
  • Microscopy, Electron
  • Rats
  • Rats, Long-Evans
  • Sensitivity and Specificity
  • Sodium Citrate
  • Solutions
  • Songbirds
  • Water


  • Antigens
  • Citrates
  • Solutions
  • Water
  • Sodium Citrate