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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\u2019s 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. 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