Journal
JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY
Volume 65, Issue 1, Pages 5-20Publisher
SAGE PUBLICATIONS LTD
DOI: 10.1369/0022155416673995
Keywords
antigen retrieval; FFPE; fixation; formalin; immunostaining
Categories
Funding
- GlaxoSmithKline
- Universita di Milano-Bicocca [HGS 1006-C1121 / BEL114054]
- Fondazione per la Ricerca Scientifica Termale (FoRST)
- MEL-PLEX research training program [642295, MSCA-ITN-2014-ETN]
- Departmental University of Milano-Bicocca and Hospital funds
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Antigen masking in routinely processed tissue is a poorly understood process caused by multiple factors. We sought to dissect the effect on antigenicity of each step of processing by using frozen sections as proxies of the whole tissue. An equivalent extent of antigen masking occurs across variable fixation times at room temperature. Most antigens benefit from longer fixation times (>24 hr) for optimal detection after antigen retrieval (AR; for example, Ki-67, bcl-2, ER). The transfer to a graded alcohol series results in an enhanced staining effect, reproduced by treating the sections with detergents, possibly because of a better access of the polymeric immunohistochemical detection system to tissue structures. A second round of masking occurs upon entering the clearing agent, mostly at the paraffin embedding step. This may depend on the non-freezable water removal. AR fully reverses the masking due both to the fixation time and the paraffin embedding. AR itself destroys some epitopes which do not survive routine processing. Processed frozen sections are a tool to investigate fixation and processing requirements for antigens in routine specimens.
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