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Dimitrov, S.* ; Shan, X.* ; Born, J. ; Inostroza, M.*

Impact of tissue storage time on immunodetection of c-Fos and GAD67 in the rat brain.

J. Neurosci. Methods 425:110602 (2025)

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BACKGROUND: Activity-dependent markers such as c-Fos, a rapid indicator of neuronal activation, and GAD67, an enzyme essential for GABA synthesis in inhibitory neurons, are extensively employed to elucidate neural circuit dynamics. Given that many studies span extended periods with multiple experimental groups, it is crucial to ensure long-term storage of non-frozen brain tissue does not compromise immunodetection. NEW METHOD: Here, we evaluated the impact of storage duration on the immunodetection of c-Fos and GAD67 in rat brains. Intact brains, fixed in paraformaldehyde, were stored at 4 °C in phosphate-buffered saline with sodium azide to prevent bacterial growth. Brains were assessed at two storage durations - short (1.5 months) and prolonged (10 months). Brain sections were immunostained for c-Fos and GAD67 and imaged by confocal microscopy. RESULTS: We observed robust c-Fos immunoreactivity across multiple regions of the mPFC, hippocampus, and neocortex, with no significant differences attributable to storage duration. Additionally, quantifications of total GAD67-positive cells and cells co-labeled for c-Fos/ GAD67 confirmed that immunodetection of inhibitory neurons remains intact when whole brains are stored for up to 10 months. In contrast, prolonged storage of brain slices strongly reduced c-Fos, but increased GAD67 staining. COMPARISON WITH EXISTING METHOD(S): The stability of c-Fos and GAD67 in tissue stored long-term at 4 °C remains untested. CONCLUSIONS: These findings underscore that whereas intact brains can be safely stored for prolonged periods at 4°C without compromising antigenicity, brain slices are highly susceptible to storage-induced deterioration - insights with implications for planning and interpreting immunohistochemical studies in neuroscience.

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