Two immunocytochemical protocols for immunofluorescent detection of c-Fos positive neurons in the rat brain

Brain Res Brain Res Protoc. 2004 Apr;13(1):45-52. doi: 10.1016/j.brainresprot.2004.01.003.

Abstract

The immediate-early-gene product c-Fos is a well known marker of neuronal activation in the central nervous system. Thus, immunocytochemical methods to detect c-Fos in the brain are important tools in experimental studies that aim to map activated brain areas on a cellular level. Accordingly, we describe here two alternative protocols for c-Fos detection which are based on an indirect immunofluorescence technique. In fact, both methods allow an excellent and specific visualisation of c-Fos immunoreactive neurons in brain areas, e.g. the hypothalamus. The first protocol is more economical and faster in its execution and useful for observing brain sections using a confocal laser scanning microscope with the intention to perform doublestaining, since in all optical magnification steps (10x-63x) only a low unspecific background staining is visible. Furthermore, this method yields even fluorescent signals which are not detectable with a conventional fluorescence-microscope at lower magnification (10x). The second protocol contains an additional signal amplification step and allows signal detection also with a conventional fluorescence-microscope at lower magnification (10x); it is useful for rapid quantification of c-Fos immunoreactive neurons in the rat brain, but because of moderate unspecific background staining at higher magnification it is less suitable for doublestaining.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Artifacts
  • Brain / cytology
  • Brain / metabolism*
  • Fluorescent Antibody Technique / methods*
  • Male
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Neurons / cytology
  • Neurons / metabolism*
  • Paraventricular Hypothalamic Nucleus / cytology
  • Paraventricular Hypothalamic Nucleus / drug effects
  • Paraventricular Hypothalamic Nucleus / metabolism
  • Proto-Oncogene Proteins c-fos / analysis*
  • Proto-Oncogene Proteins c-fos / biosynthesis
  • Rats
  • Rats, Sprague-Dawley
  • Sincalide / analogs & derivatives*
  • Sincalide / pharmacology

Substances

  • 8-sulfocholecystokinin octapeptide
  • Proto-Oncogene Proteins c-fos
  • Sincalide