RT Journal Article
SR Electronic
T1 NOT ALL ANTIBODIES HAVE BEEN CREATED EQUAL: Antibodies validated for routinely processed tissue seldom apply to frozen tissue sections
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.05.10.087361
DO 10.1101/2020.05.10.087361
A1 Maddalena M Bolognesi
A1 Francesco Mascadri
A1 Roberto Perego
A1 Silvia Bombelli
A1 Francesca M Bosisio
A1 Giorgio Cattoretti
YR 2020
UL http://biorxiv.org/content/early/2020/05/12/2020.05.10.087361.abstract
AB 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 StatementThe authors have declared no competing interest.