The effect of prolonged fixation time on immunohistochemical staining of common neurodegenerative disease markers.

The goals of this study were to determine the effects of prolonged fixation time and to optimize antigen retrieval (AR) methods on immunohistochemical (IHC) staining of common neurodegenerative disease markers. A panel of commercial antibodies (Abs) to amyloid-beta, ubiquitin, p62/sequestosome, tau, and alpha-synuclein was applied to a 2-mm tissue microarray using several AR methods. The IHC outcomes were assessed in sections that included 2 types of specimens taken from 20 postmortem brains: short-term fixation of up to 70 days before paraffin embedment and long-term fixation of up to 14 years in formalin. Good amyloid-beta IHC staining was obtained with all amyloid-beta Abs applied when a formic acid AR method was used, even after 14 years of fixation. Ubiquitin immunoreactivity was also optimally labeled with this method. The p62/sequestosome IHC outcome was optimal for tissue fixed up to 10 years, but only when the p62-lck-ligand-Ab with heat AR method was used. All hyperphosphorylated tau Abs tested worked with fixation up to 10 years, in particular with the heat AR method, whereas Abs against tau isoforms RD3 and RD4 were applicable only when the fixation time was 6 months or shorter. alpha-Synuclein-immunoreactive structures were visualized up to 14 years but only by the use of Syn42-Ab after formic acid AR or after a combination of heat and formic acid methods.