[Left] Immunofluorescent analysis of HeLa cells stained with Anti-c-Fos Antibody (A85425), dilution 1:2,000, in red, and Anti-beta Tubulin Antibody (A85428), dilution 1:10,000, in green. The blue is DAPI staining of nuclear DNA. HeLa cells were kept in fetal bovine serum (FBS)-free media for 36 hours. Then the cells were [A] treated with PBS, as a control, or [B] stimulated with 20% FBS for 30 min. Anti-c-FOS antibody labels only the nuclei of stimulated cells. [Right] [C] Mouse hippocampus or [D] olfactory bulb sections stained with Anti-c-Fos Antibody (A85425), dilution 1:10,000, in red, and Anti-NF-L Antibody (A85453), dilution 1:5,000, in green. The blue is Hoechst staining of nuclear DNA. Following transcardial perfusion of mouse with 4% paraformaldehyde, the brain was post-fixed for 24 hours, cut to 45 µm, and free-floating sections were stained with the above antibodies. The c-FOS antibody stains only nuclei of spontaneously active neurons. NF-L is expressed in axons of neuronal cells.
Western blot analysis of cell lysates using Anti-c-Fos Antibody (A85425), dilution 1:2,000, in green, and Anti-GAPDH Antibody [1D4] (A85382), dilution 1:5,000, in red, used as a loading control. The lanes contain: [1] protein standard (red), [2] HeLa cells grown in FBS free media, [3] HeLa cells stimulated with 20% FBS for 2 hours after being in FBS free media for 36 hours, [4] rat cortical neurons, and [5] rat cortical neurons treated with membrane depolarization buffer for 5 hours. Multiple bands at 50-65 kDa in stimulated or treated cell lysates correspond to different isoforms of the c-FOS protein.
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