Antigen Masking During Fixation and Embedding, Dissected

Scalia, Carla Rossana; Boi, Giovanna; Bolognesi, Maddalena M.; Riva, Lorella; Manzoni, Marco; DeSmedt, Linde; Bosisio, Francesca Maria; Ronchi, Susanna; Leone, Biagio Eugenio; Cattoretti, Giorgio

doi:10.1369/0022155416673995

Cited by 57 publications

(48 citation statements)

References 36 publications

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“…After undergoing formalin fixation, FFPE tissues are processed for paraffin embedding. This procedure involves heat-mediated solvent exchanges of ethanol, xylenes, and molten paraffin through the tissue, further washing out metabolites, lipids and molecules that may not have been crosslinked [Scalia, et al 2016]. In particular for N-glycans, which generally do not have any free amino groups and are not cross-linked, the extensive processing most likely serves to expose the N-linked glycan moieties on the remaining glycoproteins, allowing PNGaseF digestion to be more efficient.…”

Section: Methodology For N-linked Glycan Detection By Maldi Imagingmentioning

confidence: 99%

MALDI Mass Spectrometry Imaging of N-Linked Glycans in Cancer Tissues

Drake

Powers

Jones

et al. 2017

Advances in Cancer Research

129

112

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Glycosylated proteins account for a majority of the post-translation modifications of cell surface, secreted and circulating proteins. Within the tumor microenvironment, the presence of immune cells, extracellular matrix proteins, cell surface receptors and interactions between stroma and tumor cells are all processes mediated by glycan binding and recognition reactions. Changes in glycosylation during tumorigenesis are well documented to occur, and affect all of these associated adhesion and regulatory functions. A MALDI imaging mass spectrometry (MALDI-IMS) workflow for profiling N-linked glycan distributions in fresh/frozen tissues and formalin-fixed paraffin-embedded (FFPE) tissues has recently been developed. The key to the approach is the application of a molecular coating of peptide-N-glycosidase to tissues, an enzyme that cleaves asparagine-linked glycans from their protein carrier. The released N-linked glycans can then be analyzed by MALDI-IMS directly on tissue. Generally 40 or more individual glycan structures are routinely detected, and when combined with histopathology localizations, tumor specific glycans are readily grouped relative to non-tumor regions and other structural features. This technique is a recent development and new approach in glycobiology and mass spectrometry imaging research methodology, thus potential uses such as tumor-specific glycan biomarker panels and other applications are discussed.

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Section: Methodology For N-linked Glycan Detection By Maldi Imagingmentioning

confidence: 99%

MALDI Mass Spectrometry Imaging of N-Linked Glycans in Cancer Tissues

Drake

Powers

Jones

et al. 2017

Advances in Cancer Research

129

112

View full text Add to dashboard Cite

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“…When compared to the mRNA-based ER pathway activity assay, the dual staining ER activity assay has the advantage of being able to assess ER activity within preserved tissue architecture, but it lacks the quantitative nature of the mRNA-based assay. It needs to be explored to what extent the assay is compatible with other types of sample preparation, such as formalin fixated paraffin embedded tissue 13 . The presented staining assay is expected to have potential value, complementary to the mRNA-based ER pathway activity assay.…”

Section: Discussionmentioning

confidence: 99%

A novel dual antibody staining assay to measure estrogen receptor transcriptional activity

Hemert¹,

Veen²,

Konings³

et al. 2020

Preprint

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Activity of the canonical estrogen receptor (ER) pathway is equivalent to functional activity of the nuclear ER transcription factor. To assess transcriptional activity of ER, for biomedical research and diagnostic purposes ER monoclonal antibodies are routinely used to identify nuclear ER staining in cells and tissue samples, however it remained unclear whether this is sufficiently predictive for transcriptional activity of ER, and thus for ER pathway activity. Using ER positive breast cancer cell lines (MCF7 and T47D) in which the transcriptional activity status of ER was quantified using an mRNA based ER pathway activity assay, the relation between ER activity and nuclear ER staining with ER monoclonal antibodies (MoAb) was investigated. While the presence of ER in the cell nucleus is a prerequisite for ER activity, it was not predictive for ER transcriptional activity, confirming earlier findings. There were remarkable differences in behaviour of the used MoAbs: EP1 and 1D5 MoAbs showed reduced nuclear staining when ER was transcriptionally active, while staining with H4624 MoAb was independent of ER activity. To improve discrimination between active and inactive nuclear ER based on ER staining, a method was developed which consists of dual ER MoAb immunofluorescent staining, followed by generation of a digital image with a standard digital pathology scanner, and application of a cell nucleus detection algorithm and per cell calculation of the nuclear H4624/EP1 fluorescence intensity ratio, where a high H4624/EP1 ratio predicts an active ER. In this method the EP1 MoAb can in principle be replaced by the 1D5 MoAb. We hypothesize that the EP1 and 1D5 monoclonal antibodies (MoAb) recognize an ER epitope which becomes hidden upon transcriptional activation of ER, while the H4624 MoAb binds an ER epitope which remains accessible when ER is activated. The method is expected to be of value to better assess ER activity by means of staining.

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“…On the other hand, the type of tissue target in Surgical Pathology is overwhelmingly FFPE tissue sections, whose pre-analytical variables are also highly regulated (8). The modifications of the protein targets in FFPE material and the performance of antibodies in such conditions have been extensively published and are the basis for a successful use of FFPE tissue for diagnostic, prognostic and theragnostic purposes (9)(10)(11). Because of the robustness of tissue processing and immunoanalysis, archived FFPE human material and Surgical Pathology techniques are also the most abundant and diffuse tools for translational and basic science research.…”

Section: Introductionmentioning

confidence: 99%

NOT ALL ANTIBODIES HAVE BEEN CREATED EQUAL: Antibodies validated for routinely processed tissue seldom apply to frozen tissue sections

Bolognesi

Mascadri

Perego

et al. 2020

Preprint

Self Cite

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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, paraffinembedded (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.

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Antigen Masking During Fixation and Embedding, Dissected

Cited by 57 publications

References 36 publications

MALDI Mass Spectrometry Imaging of N-Linked Glycans in Cancer Tissues

MALDI Mass Spectrometry Imaging of N-Linked Glycans in Cancer Tissues

A novel dual antibody staining assay to measure estrogen receptor transcriptional activity

NOT ALL ANTIBODIES HAVE BEEN CREATED EQUAL: Antibodies validated for routinely processed tissue seldom apply to frozen tissue sections

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