Formalin-fixed, paraffin-embedded (FFPE) tissues have been widely used in researches. Proteins and nucleic acids in prolonged FFPE tissues display different degrees of degradation. We investigated the effect of prolonged formalin fixation on protein expression in human brain tissues. Twenty-eight middle prefrontal front cortex tissue blocks from human brains prefixed in formalin were obtained from a brain bank. The tissue blocks were divided into two groups, the control group and the prolonged fixation group. Quantitative immunocytochemistry was used to analyse the biological markers of Fox-3, Rbfox3 (NeuN), glial fibrillary acidic protein (GFAP), ionized calcium binding adapter molecule-1 (IBA-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Nissl staining showed that positive signaling of Nissl body was significantly decreased by 16.6% in the prolonged fixation group. In addition, the staining intensity of Nissl body was negatively correlated with fixation time. The level of NeuN immunoreactivity (ir) was significantly reduced by 19.31% in the prolonged fixation group. Moreover, there was a significant negative correlation between NeuN-ir and fixation time. There were no significant changes in GFAP-ir, IBA-1-ir and GAPDH-ir between control group and the prolonged fixation group. Prolonged formalin-fixed tissues showed time- and molecule-dependent protein changes, which may be potential confounders in the clinic and researches. Our study suggested short formalin fixation time is recommended when using PPFE brain tissues.
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