Effects of prolonged fixation on vascular biomarkers in postmortem human brains

Postmortem human brains are important research resources to study the mechanisms underlying cerebrovascular features in various neurodegenerative disorders. Immunohistochemical and histochemical staining have been used to examine human brains fixed in neutral-buffered formalin (NBF) for months, years, or decades. Previously, we found that prolonged NBF fixation resulted in differential effects on immunohistochemistry and histochemistry staining on postmortem brains. Here, we further examined the effects of prolonged fixation (1, 5, 10, 15, and 20 years) on stains of known biomarkers of cerebrovascular diseases in the human prefrontal cortex. We included microvasculature markers of the blood vessel wall (anti-collagen-IV and claudin-5), a type III intermediate filament marker (anti-vimentin), an activated microglia marker (anti-CD68), a biomarker for proteolipid protein (anti-PLP) of oligodendrocytes and a marker for iron accumulation (anti-ferritin). We also included Masson's trichrome stain (MTS) and Bielschowsky silver stain (BSS). We found that staining intensities of ferritin, vimentin, collagen-IV, and BSS decreased with prolonged fixation. No significant differences were observed in the staining intensity of other markers. We therefore recommend performing IHC and HC staining for human brains with the same fixation times to offset any impact on downstream neuropathological analyses, as well as adding the fixation duration as a covariate in the analysis.