A Simple Method for Immunohistochemical Staining of Zebrafish Brain Sections for c-fos Protein Expression

Immediate early genes (IEGs) are transcription factors whose own transcription is initiated rapidly, for example, in the brain in response to environmental stimuli. c-fos is an IEG often used as a marker of neuronal activation. c-fos mRNA expression has started to be quantified and localized in the zebrafish brain following environmental manipulations but analysis of the expression of c-fos protein in the zebrafish brain has rarely been attempted. Here, we describe an immunofluorescence staining method for quantifying c-fos protein expression in different regions of the zebrafish brain. In addition, we expose zebrafish to caffeine, a positive control for c-fos activation in the brain. To confirm cell nucleus specific binding of the c-fos antibody, we counterstained brain sections with the nuclear fluorescent stain DAPI. Furthermore, we describe a method for reducing background autofluorescence often observed in zebrafish brain tissue. Our analysis showed that exposure to caffeine increased the number of c-fos protein-positive cells in specific zebrafish brain regions detected by the immunofluorescence method. Our results demonstrate the feasibility of immunofluorescence-based methods in the analysis of neuronal activation in the zebrafish brain, and reinforce the utility of the zebrafish in behavioral neuroscience research.