ArticleModern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc2016
Multilabel immunofluorescence and antigen reprobing on formalin-fixed paraffin-embedded sections: novel applications for precision pathology diagnosis.
Article in Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 11 papers.
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11 citing papers in PubMed, 18 citations in OpenAlex.
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- Novel CARMIL2 loss-of-function variants are associated with pediatric inflammatory bowel disease.Scientific reports · 2021Article
- A Straightforward Method for 3D Visualization of B Cell Clusters and High Endothelial Venules in Lymph Nodes Highlights Differential Roles of TNFRI and -II.Frontiers in immunology · 2021Article
- Subcellular proteomics.Nature reviews. Methods primers · 2021Article
- The E3 ubiquitin ligase UBR5 interacts with TTC7A and may be associated with very early onset inflammatory bowel disease.Scientific reports · 2020Article
- Multiplex Immunofluorescence of Bone Marrow Core Biopsies: Visualizing the Bone Marrow Immune Contexture.The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society · 2020Article
- Expression Analysis of ATP-Binding Cassette Transporters ABCB11 and ABCB4 in Primary Sclerosing Cholangitis and Variety of Pediatric and Adult Cholestatic and Noncholestatic Liver Diseases.Canadian journal of gastroenterology & hepatology · 2019Article
- Mind the Gaps in Tumor Immunity: Impact of Connexin-Mediated Intercellular Connections.Frontiers in immunology · 2017Review
- Hyperplasia and hypertrophy of pulmonary neuroepithelial bodies, presumed airway hypoxia sensors, in hypoxia-inducible factor prolyl hydroxylase-deficient mice.Hypoxia (Auckland, N.Z.) · 2016Article
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
Abstract
We report new methods for multilabel immunofluorescence (MIF) and reprobing of antigen epitopes on the same formalin-fixed paraffin-embedded (FFPE) sections. The MIF method includes an antigen-retrieval step followed by multilabel immunostaining and examination by confocal microscopy. As examples, we illustrate epitopes localized to the apical and basolateral membranes, and the cytoplasm of enterocytes of normal small intestine and in cases of congenital enteropathies (microvillous inclusion disease and congenital tufting enteropathy). We also demonstrate localization of the bile salt excretion pump protein (BSEP) in bile canalicular membrane of normal hepatocytes and in cases of primary sclerosing cholangitis. To demonstrate colocalization of cytoplasmic and nuclear epitopes we analyzed normal control and hyperplastic pulmonary neuroendocrine cells (PNEC) and neuroepithelial bodies (NEBs), presumed airway sensors in the lungs of infants with bronchopulmonary dysplasia (BPD). As cytoplasmic markers we used anti-bombesin or anti-synaptic vesicle protein 2 (SV2) antibody, respectively, and for nuclear localization, antibodies against neurogenic genes mammalian achaete-scute homolog (Mash1) and prospero homeobox 1 (Prox1), essential for NEB cells differentiation and maturation, hypoxia-inducible factor 1α (HIF1α) a downstream modulator of hypoxia response and a proliferation marker Ki67. The reprobing method consisted of removal of the previously immunolabeled target and immunostaining with different antibodies, facilitating colocalization of enterocyte brush border epitopes as well as HIF1α, Mash1 and Prox1 in PNEC/NEB PNEC and NEBs. As these methods are suitable for routine FFPE pathology samples from various tissues, allowing visualization of multiple epitopes in the same cells/sections with superior contrast and resolution, they are suitable for a wide range of applications in diagnostic pathology and may be particularly well suited for precision medicine diagnostics.
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