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 (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.
Kobelt
Peter
P
Department of Medicine, Division Hepatology and Gastroenterology, Charité, Campus Virchow-Klinikum, Humboldt-Universität Berlin, Germany.
Tebbe
Johannes J
JJ
Tjandra
Ines
I
Bae
Hi-Gung
HG
Rüter
Jens
J
Klapp
Burghard F
BF
Wiedenmann
Bertram
B
Mönnikes
Hubert
H
D000818:Animals; D016477:Artifacts; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique / Q000379:methods*; D008297:Male; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D010286:Paraventricular Hypothalamic Nucleus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000032:analysis* / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D012844:Sincalide / Q000031:analogs & derivatives* / Q000494:pharmacology
C090019:8-sulfocholecystokinin octapeptide; D016760:Proto-Oncogene Proteins c-fos; D012844:Sincalide