Pre-embedding immunolabeling for electron microscopy: An evaluation of permeabilization methods and markers

Humbel, Bruno M.; Jong, Margo D.M. de; Müller, Wally H.; Verkleij, Arie J.

doi:10.1002/(sici)1097-0029(19980701)42:1<43::aid-jemt6>3.0.co;2-s

Cited by 77 publications

(29 citation statements)

References 54 publications

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“…Colloidal gold is classically used; this bulky tag, however, often results in low-level staining and significant background, presumably arising from the negative charge and poor penetration of the bulky colloidal gold (Humbel et al 1998). Recent studies illustrated improved properties, with better penetration, of the commercial ABs called FluoroNanogold (Nanoprobes).…”

Section: Fluoronanogold Secondary Absmentioning

confidence: 99%

Chromosome structure: improved immunolabeling for electron microscopy

2005

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To structurally dissect mitotic chromosomes, we aim to position along the folded chromatin fiber proteins involved in long-range order, such as topoisomerase IIalpha (topoIIalpha) and condensin. Immuno-electron microscopy (EM) of thin-sectioned chromosomes is the method of choice toward this goal. A much-improved immunoprocedure that avoids problems associated with aldehyde fixation, such as chemical translinking and networking of chromatin fibers, is reported here. We show that ultraviolet irradiation of isolated nuclei or chromosomes facilitates high-level specific immunostaining, as established by fluorescence microscopy with a variety of antibodies and especially by immuno-EM. Ultrastructural localizations of topoIIalpha and condensin I component hBarren (hBar; hCAP-H) in mitotic chromosomes were studied by immuno-EM. We show that the micrographs of thin-sectioned chromosomes map topoIIalpha and hBar to the center of the chromosomal body where the chromatin fibers generally converge. This localization is defined by many clustered gold particles with only rare individual particles in the peripheral halo. The data obtained are consistent with the view that condensin and perhaps topoIIalpha tether chromatin to loops according to a scaffolding-type model.

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Section: Fluoronanogold Secondary Absmentioning

confidence: 99%

Chromosome structure: improved immunolabeling for electron microscopy

2005

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“…Nevertheless, glutaraldehyde is popular because it gives superior preservation of tissue morphology. Similarly, dehydration, defatting and embedding also affect antibody penetration and will cause both masking and unmasking of epitopes which may or may not be of relevance for the specificity or sensitivity of the overall procedure (e.g., Pool and Buijs 1988;Humbel et al 1998).…”

Section: Introductionmentioning

confidence: 99%

Specificity controls for immunocytochemistry

2006

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Antibodies have been in widespread use for more than three decades as invaluable tools for the specific detection of proteins or other molecules in biological samples. In spite of such a long experience, the field of immunocytochemistry is still troubled by spurious results due to insufficient specificity of antibodies or procedures used. The importance of keeping a high standard is increasing because massive sequencing of entire genomes leads to the identification of numerous new proteins. All the identified proteins and their variants will have to be localized precisely and quantitatively at high resolution throughout the development of all species. Consequently, antibody generation and immunocytochemical investigations will be done on a large scale. It will be economically important to secure an optimal balance between the risk of publishing erroneous data (which are expensive to correct) and the costs of specificity testing. Because proofs of specificity are never absolute, but rather represent failures to detect crossreactivity, there is no limit to the number of control experiments that can be performed. The aims of the present paper are to increase the awareness of the difficulties in proving the specificity of immunocytochemical labeling and to initiate a discussion on optimized standards. The main points are: (1) antibodies should be described properly, (2) the labeling obtained with an antibody to a single epitope needs additional verification and (3) the investigators should be required to outline in detail how they arrive at the conclusion that the immunocytochemical labeling is specific.

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“…Virtual pre-embedding procedure With virtual pre-embedding (VirP), haptens are used in a combination of pre-and post-embedding procedures (Humbel et al 1998). Haptens are deposited in close vicinity of the antigen by catalysed reporter deposition (CARD) in a pre-embedding step (Bobrow et al 1989).…”

Section: Preparation Of Tetramethylrhodamine-tyramidementioning

confidence: 99%

Synaptic localisation of agmatinase in rat cerebral cortex revealed by virtual pre-embedding

et al. 2011

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Light microscopic evidence suggested a synaptic role for agmatinase, an enzyme capable of inactivating the putative neurotransmitter and endogenous anti-depressant agmatine. Using electron microscopy and an alternative pre-embedding approach referred to as virtual pre-embedding, agmatinase was localised pre- and postsynaptically, to dendritic spines, spine and non-spine terminals, and dendritic profiles. In dendritic spines, labelling displayed a tendency towards the postsynaptic density. These results further strengthen a synaptic role for agmatine and strongly suggest a regulatory role for synaptically expressed agmatinase.

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Pre-embedding immunolabeling for electron microscopy: An evaluation of permeabilization methods and markers

Cited by 77 publications

References 54 publications

Chromosome structure: improved immunolabeling for electron microscopy

Chromosome structure: improved immunolabeling for electron microscopy

Specificity controls for immunocytochemistry

Synaptic localisation of agmatinase in rat cerebral cortex revealed by virtual pre-embedding

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