Antibodies Validated for Routinely Processed Tissues Stain Frozen Sections Unpredictably

Bolognesi, Maddalena M.; Mascadri, Francesco; Furia, Laura; Faretta, Mario; Bosisio, Francesca Maria; Cattoretti, Giorgio

doi:10.2144/btn-2020-0149

Cited by 6 publications

(8 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A pilot study on colon adenocarcinomas (COADs) employing frozen tissue sections, was run with a limited, one‐shot multiplex combinations directed at DDR molecules, with antibodies compatible with frozen material 16 (Table S1). It showed two distinct staining patterns: on one side tumors with rare, mostly isolated γH2AX+ nuclei and absence of pNBS1 and pCHK2; on the other side, neoplastic glands containing γH2AX, pNBS1, and pCHK2, nonproliferating (Figure S4).…”

Section: Resultsmentioning

confidence: 99%

“…A pilot study involved matched cryosections from isopentane‐frozen tissue at −80°C and sections from routinely processed tissue (formalin‐fixed, paraffin embedded; FFPE) from a small number of consecutive anonymous cases, with the scope of validating the staining conditions for both as reported previously 16 . The multiplexing MILAN technique 17 (see below) is not applicable to frozen sections.…”

Section: Methodsmentioning

confidence: 99%

“…Primary unconjugated antibodies were validated for frozen and for FFPE material according to established criteria 16 (see Table S1).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

A DNA damage response‐like phenotype defines a third of colon cancers at onset

et al. 2023

Self Cite

View full text Add to dashboard Cite

Colon adenocarcinoma (COAD) has a limited range of diversified, personalized therapeutic opportunities, besides DNA hypermutating cases; thus, both new targets or broadening existing strategies for personalized intervention are of interest. Routinely processed material from 246 untreated COADs with clinical follow‐up was probed for evidence of DNA damage response (DDR), that is, the gathering of DDR‐associated molecules at discrete nuclear spots, by multiplex immunofluorescence and immunohistochemical staining for DDR complex proteins (γH2AX, pCHK2, and pNBS1). We also tested the cases for type I interferon response, T‐lymphocyte infiltration (TILs), and mutation mismatch repair defects (MMRd), known to be associated with defects of DNA repair. FISH analysis for chromosome 20q copy number variations was obtained. A total of 33.7% of COAD display a coordinated DDR on quiescent, non‐senescent, non‐apoptotic glands, irrespective of TP53 status, chromosome 20q abnormalities, and type I IFN response. Clinicopathological parameters did not differentiate DDR+ cases from the other cases. TILs were equally present in DDR and non‐DDR cases. DDR+ MMRd cases were preferentially retaining wild‐type MLH1. The outcome after 5FU‐based chemotherapy was not different in the two groups. DDR+ COAD represents a subgroup not aligned with known diagnostic, prognostic, or therapeutic categories, with potential new targeted treatment opportunities, exploiting the DNA damage repair pathways.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

A DNA damage response‐like phenotype defines a third of colon cancers at onset

et al. 2023

Self Cite

View full text Add to dashboard Cite

show abstract

“…11 Disaccharides such as lactose or sucrose can help in avoiding these negative effects by acting as molecular water substitutes and/or protein conformation facilitators. 9 Finally, formalin-induced bonds formed after 48 hr of fixation of frozen sections from fresh tissue can be reversed completely by as little as 20 min of AR, leading to tissue loss, 12 which do not occur in routinely processed tissue (FFPE) fixed in formalin for shorter time.…”

Section: Introductionmentioning

confidence: 99%

“…Finally, formalin-induced bonds formed after 48 hr of fixation of frozen sections from fresh tissue can be reversed completely by as little as 20 min of AR, leading to tissue loss, 12 which do not occur in routinely processed tissue (FFPE) fixed in formalin for shorter time.…”

Section: Introductionmentioning

confidence: 99%

Rejuvenated Vintage Tissue Sections Highlight Individual Antigen Fate During Processing and Long-term Storage

Mascadri

Bolognesi

Pilla

et al. 2021

J Histochem Cytochem.

Self Cite

View full text Add to dashboard Cite

Antigen-bearing proteins become progressively unavailable to immunodetection after prolonged storage of routine sections, exposed to a variety of agents, such as moisture, oxygen, and temperature. By proteomic analysis, the antigens are retained in the sections and definitely in the tissue block, pointing to fixation-independent, storage time–dependent protein modifications. Based on previous experience, we hypothesized that a combined exposure to a reducing agent and to chemicals favoring protein conformation changes would reverse the masking in aged sections. Disaccharides, lactose and sucrose, and a surfactant, added to a standard antigen retrieval buffer, reverse the negative changes in aged sections. Furthermore, they provide enhanced access to antigens in freshly cut sections, but not universally, revealing additional factors, besides heat and calcium chelation, required for antigen retrieval of individual proteins:

show abstract

Seeing or believing in hyperplexed spatial proteomics via antibodies: New and old biases for an image-based technology

Bolognesi,

Dall’Olio,

Maerten

et al. 2024

Biol. Imaging

View full text Add to dashboard Cite

Hyperplexed in-situ targeted proteomics via antibody immunodetection (i.e., >15 markers) is changing how we classify cells and tissues. Differently from other high-dimensional single-cell assays (flow cytometry, single-cell RNA sequencing), the human eye is a necessary component in multiple procedural steps: image segmentation, signal thresholding, antibody validation, and iconographic rendering. Established methods complement the human image evaluation, but may carry undisclosed biases in such a new context, therefore we re-evaluate all the steps in hyperplexed proteomics. We found that the human eye can discriminate less than 64 out of 256 gray levels and has limitations in discriminating luminance levels in conventional histology images. Furthermore, only images containing visible signals are selected and eye-guided digital thresholding separates signal from noise. BRAQUE, a hyperplexed proteomic tool, can extract, in a marker-agnostic fashion, granular information from markers which have a very low signal-to-noise ratio and therefore are not visualized by traditional visual rendering. By analyzing a public human lymph node dataset, we also found unpredicted staining results by validated antibodies, which highlight the need to upgrade the definition of antibody specificity in hyperplexed immunostaining. Spatially hyperplexed methods upgrade and supplant traditional image-based analysis of tissue immunostaining, beyond the human eye contribution.

show abstract

Antibodies Validated for Routinely Processed Tissues Stain Frozen Sections Unpredictably

Cited by 6 publications

References 35 publications

A DNA damage response‐like phenotype defines a third of colon cancers at onset

A DNA damage response‐like phenotype defines a third of colon cancers at onset

Rejuvenated Vintage Tissue Sections Highlight Individual Antigen Fate During Processing and Long-term Storage

Seeing or believing in hyperplexed spatial proteomics via antibodies: New and old biases for an image-based technology

Contact Info

Product

Resources

About