Differential effects of prolonged post-fixation on immunohistochemical and histochemical staining for postmortem human brains

Immunohistochemical (IHC) and histochemical (HC) staining is widely used for human brains post-fixed in formalin and stored in brain banks worldwide for months, years and decades. Understanding the effect of prolonged post-fixation, postmortem interval (PMI) and age on these staining procedures is important for interpreting their outcomes, thus improving diagnosis and research of brain disorders afflicting millions of world populations. In this study, we performed both IHC and HC staining for prefrontal cortex (PFC) of postmortem human brains post-fixed for 1, 5, 10, 15 and 20 years. A negative correlation was detected between the intensity of neuron marker neuron nuclear specific marker (NeuN), microglia marker ionized calcium-binding adaptor molecule 1 (Iba1), cresyl violet (CV) and Luxol fast blue (LFB) staining versus post-fixation durations. By contrast, a positive correlation was seen between the intensity of astrocyte marker glial fibrillary acidic protein (GFAP) and hemaetoxylin and eosin Y (H&E) staining versus post-fixation durations. No correlation was found between the staining intensity of NeuN, GFAP, Iba1, H&E, CV and LFB versus PMI. Moreover, no correlation was seen between NeuN, Iba1, H&E, CV and LFB, except GFAP, versus age. These data suggest that prolonged post-fixation exerts both positive and negative effects, but age and PMI have limited effects, on these IHC and HC parameters. Hence these differential changes need to be considered in interpretation of the results when using tissues with prolonged post-fixation. Furthermore, if feasible, it is recommended to perform IHC and HC staining for human brains with the same post-fixation time windows and to use the most optimal antibodies to offset its impact on downstream analyses.