Autofluorescent cells in rat brain can be convincing impostors in green fluorescent reporter studies

Cell transplant and gene therapies are promising approaches to many disorders of the nervous system. In studies involving cell transplants to the brain or nervous system, expression of green fluorescent protein (GFP) is commonly used to label cells, allowing their identification and histological assessment even after long post-operative survival times. Techniques employing viral tracing or reporter genes also commonly use GFP to label cells. Here, we document the presence of a subpopulation of green autofluorescent cells in the cortex and hippocampus of formaldehyde fixed, cryosectioned rat brains aged 3-9 months. Using standard microscopic fluorescence imaging techniques, we acquired clear images of green autofluorescent cells, complete with extensive processes, which appear to be well integrated into the host tissue. Treatment of brain sections with sodium borohydride followed by cupric sulfate in ammonium acetate buffer reduced background and cellular autofluorescence throughout sections but, especially in hippocampus, did not eliminate considerable green fluorescence in a subset of neurons. This autofluorescence was weak and would therefore pose a problem only when cells weakly express GFP or when few labeled cells survive. We suggest that investigators be aware of the potential for false positives, especially if the cells expressing GFP are expected to migrate widely from the transplant site. Parallel sections from naïve brains should regularly be processed and imaged alongside experimental brain sections, and anti-GFP immunohistochemistry should be performed to ensure that true GFP+ signals are imaged instead of endogenous autofluorescent neurons.