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

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 (10×–63×) 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 (10×). The second protocol contains an additional signal amplification step and allows signal detection also with a conventional fluorescence-microscope at lower magnification (10×); 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.