Amplification Methods for the Immunolocalizationof Rare Molecules in Cells and Tissues

Mayer, Gaétan; Bendayan, Moı̈se

doi:10.1016/s0079-6336(01)80002-4

Cited by 20 publications

(11 citation statements)

References 318 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Steric constraints are then likely to dictate the angle and preferential orientation by which some antibodies bind to certain NPC components. In fact, when using IgGs as primary antibodies, together with gold-coupled secondary antibodies for indirect immunolabelings, the mean distance between target and peak distribution of gold particles can come close to 30 nm (Kellenberger and Hayat, 1991;Mayer and Bendayan, 2001). In contrast, antibodies binding to epitopes only exposed on the surface of ultrathin sections through resin-embedded NPCs can enjoy more rotational freedom.…”

Section: Resultsmentioning

confidence: 99%

Nucleoporins as Components of the Nuclear Pore Complex Core Structure and Tpr as the Architectural Element of the Nuclear Basket

Krull

Thyberg

Björkroth

et al. 2004

MBoC

214

233

View full text Add to dashboard Cite

The vertebrate nuclear pore complex (NPC) is a macromolecular assembly of protein subcomplexes forming a structure of eightfold radial symmetry. The NPC core consists of globular subunits sandwiched between two coaxial ring-like structures of which the ring facing the nuclear interior is capped by a fibrous structure called the nuclear basket. By postembedding immunoelectron microscopy, we have mapped the positions of several human NPC proteins relative to the NPC core and its associated basket, including Nup93, Nup96, Nup98, Nup107, Nup153, Nup205, and the coiled coil-dominated 267-kDa protein Tpr. To further assess their contributions to NPC and basket architecture, the genes encoding Nup93, Nup96, Nup107, and Nup205 were posttranscriptionally silenced by RNA interference (RNAi) in HeLa cells, complementing recent RNAi experiments on Nup153 and Tpr. We show that Nup96 and Nup107 are core elements of the NPC proper that are essential for NPC assembly and docking of Nup153 and Tpr to the NPC. Nup93 and Nup205 are other NPC core elements that are important for long-term maintenance of NPCs but initially dispensable for the anchoring of Nup153 and Tpr. Immunogold-labeling for Nup98 also results in preferential labeling of NPC core regions, whereas Nup153 is shown to bind via its amino-terminal domain to the nuclear coaxial ring linking the NPC core structures and Tpr. The position of Tpr in turn is shown to coincide with that of the nuclear basket, with different Tpr protein domains corresponding to distinct basket segments. We propose a model in which Tpr constitutes the central architectural element that forms the scaffold of the nuclear basket.

show abstract

Section: Resultsmentioning

confidence: 99%

Nucleoporins as Components of the Nuclear Pore Complex Core Structure and Tpr as the Architectural Element of the Nuclear Basket

Krull

Thyberg

Björkroth

et al. 2004

MBoC

214

233

View full text Add to dashboard Cite

show abstract

“…Despite continuous efforts to improve the sensitivity and specificity of MS and IHC, for example the introduction of nanoparticles 3, 4, 16 in combination with signal amplification techniques 17, many protein species in a tissue section remain difficult to detect. At present, the only tool available that provides in‐depth proteomic measurements while preserving two‐dimensional architecture is tissue microdissection, which procures specific cells from a histological section for molecular analysis 18–20.…”

Section: Discussionmentioning

confidence: 99%

Layered electrophoretic transfer – A method for pre‐analytic processing of histological sections

et al. 2011

View full text Add to dashboard Cite

Current technologies for measuring protein expression across a tissue section are based on mass spectrometry or in situ detection such as immunohistochemistry. However, due to the inherent molecular complexity of tissue samples and the large dynamic range of protein expression in cells, current approaches are often unable to measure moderate- and low-abundant proteins. In addition, they do not provide information on the physico- chemical properties of the proteins studied. To address these problems, we are developing a new pre-analytic methodology termed Layered Electrophoretic Transfer (LET) that selectively separates and processes proteins from an intact tissue section without compromising important two-dimensional histological information. LET offers two potential advantages over standard techniques: 1) A reduced complexity of the tissue proteome for subsequent analysis; 2) An opportunity to assess the biochemical status of proteins as they exist in situ. As an initial proof-of-concept, we demonstrate here that the protein content from a mixture of molecular weight standards, human tissue lysates, and tissue sections can be successfully transferred and separated using LET, and further demonstrate that the method can be coupled with immunoblotting or mass spectrometry for downstream measurements. LET technology represents a new pre-analytic tool for interrogating the proteome in tissue sections while preserving valuable spatial information.

show abstract

“…An already available example was the introduction of the reactions performed in a single stage (EPOS) 127. This involves an inert polymer in which many molecules from the primary antibody and peroxidase are chemically connected, consequently decreasing the number of incubation stages, and is currently commercially available.…”

Section: Quantification Of Immunohistochemistry Expressionmentioning

confidence: 99%

Immunohistochemistry as an Important Tool in Biomarkers Detection and Clinical Practice

et al. 2010

View full text Add to dashboard Cite

The immunohistochemistry technique is used in the search for cell or tissue antigens that range from amino acids and proteins to infectious agents and specific cellular populations. The technique comprises two phases: (1) slides preparation and stages involved for the reaction; (2) interpretation and quantification of the obtained expression. Immunohistochemistry is an important tool for scientific research and also a complementary technique for the elucidation of differential diagnoses which are not determinable by conventional analysis with hematoxylin and eosin. In the last couple of decades there has been an exponential increase in publications on immunohistochemistry and immunocytochemistry techniques. This review covers the immunohistochemistry technique; its history, applications, importance, limitations, difficulties, problems and some aspects related to results interpretation and quantification. Future developments on the immunohistochemistry technique and its expression quantification should not be disseminated in two languages—that of the pathologist and another of clinician or surgeon. The scientific, diagnostic and prognostic applications of this methodology must be explored in a bid to benefit of patient. In order to achieve this goal a collaboration and pooling of knowledge from both of these valuable medical areas is vital

show abstract

Amplification Methods for the Immunolocalizationof Rare Molecules in Cells and Tissues

Cited by 20 publications

References 318 publications

Nucleoporins as Components of the Nuclear Pore Complex Core Structure and Tpr as the Architectural Element of the Nuclear Basket

Nucleoporins as Components of the Nuclear Pore Complex Core Structure and Tpr as the Architectural Element of the Nuclear Basket

Layered electrophoretic transfer – A method for pre‐analytic processing of histological sections

Immunohistochemistry as an Important Tool in Biomarkers Detection and Clinical Practice

Contact Info

Product

Resources

About