Analysis of c-Fos expression in cultured HeLa cells by Immunocytochemistry (A-B), and in mouse hippocampus (C) or olfactory bulb sections (D) by Immunohistochemistry. HeLa cells were kept in serum-free medium for 36 hours, before cells were stimulated with 20% FBS for 30 minutes (B), while control cells (A) were treated with PBS as a control. The rabbit anti-c-Fos antibody (red, 1:5,000) labels nuclei of stimulated cells, while DAPI (blue) stains all cell nuclei independent of their activity stage. Green: mouse anti-tubulin antibody. Mouse hippocampus (C) and olfactory bulb sections (D) were stained with rabbit anti-c-Fos antibody (red, 1:20,000) and mouse antibody to NF-L (green). The c-FOS antibody stains only nuclei of spontaneously active neurons. NF-L is expressed in axons of neuronal cells. Blue: Cell nuclei stained with DAPI. IHC Method: Following transcardial perfusion of mouse with 4% paraformaldehyde, brain was post fixed for 24 hours, cut to 45 uM, and free-floating sections were stained with above antibodies.