ABSTRACT
A customized context-dependent validation of antibodies for the prospective use, rather than a general stamp of validity, is required for reproducibility and data validity, besides the need of standardized reagents. In-situ antibody staining is a technique broadly used in experimental settings and human diagnostic practice. The first typically, but not exclusively uses lightly fixed material (cell smears, frozen sections), the second, routinely processed formalin-fixed, paraffin-embedded (FFPE) tissue. Differently from techniques based on tissue extraction, there is little awareness of the context-dependent constraints inherent with either type of in situ staining except that antigen masking associated with routine tissue processing limits the range of useful antibodies. We applied a panel of 126 antibodies validated for FFPE to lightly fixed (acetone, formalin) frozen sections and found that less than 30% performed conservatively with all fixations, 35% preferred one fixation over another, 13% gave non-specific staining, 23% did not stain at all. Individual antibody variegation of the paratope fitness for the differentially fixed antigen may be the cause. Re-validation of established antibody panels is required when applied to sections whose fixation and processing is different from the tissue where they have been initially validated.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
FINANCIAL SUPPORT: This work has been supported by Departmental University of Milano-Bicocca funds and by a Fondazione Cariplo grant (2017-0577) to Prof. Roberto Perego. MMB is a PhD student in the DIMET program..
Mailing address: Prof. Giorgio Cattoretti, Anatomia Patologica, UNIMIB and Ospedale San Gerardo, Via Pergolesi 33, 20900, Monza (IT). Phone: +39 039-233-2551 (office); +30 02-6448-8068 (lab); FAX: +39 039-233-2548; e-mail: giorgio.cattoretti{at}unimib.it
CONFLICT OF INTERESTS: The Authors have no conflict of interests to declare.