Immunolabeling with the Protein A-Gold Technique: An Overview

Roth, Jürgen; Heitz, Philipp U.

doi:10.3109/01913128909074531

Cited by 27 publications

(15 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In B cells immunostained with protein gold-silver enhancement at the LM level, gold-silver particles labeling insulin in the cores of secretory granules were revealed by the IE-immunoSEM technique; these findings were consistent with results obtained from immu-noTEM studies (Bendayan, 1984(Bendayan, , 1989(Bendayan, , 1993Bendayan and Garzon, 1988;Lee, 2003;Orci et al, 1984Orci et al, , 1987Roth and Heitz, 1989;Roth et al, 1978Roth et al, , 1996. The fragile structure of organelles such as endocrine secretory granules was clarified by mild IE.…”

Section: Discussionsupporting

confidence: 87%

“…Immunolabeling methodology employed in the present investigation consists of the protein gold technique (Bendayan, 1984) with silver enhancement, which has been applied in both light (Danscher and Norgaard, 1983;Nanci et al, 1990;Piludu et al, 2003;Roth and Heitz, 1989;Roth et al, 1996) and scanning electron microscopic immunocytochemistry (Bendayan, 2000;Carrassi et al, 1990;Nanci et al, 1990;Scopsi et al, 1986). Roth and Heitz (1989) noted that gold particles as small as 5 nm may be used for light microscopic labeling. Small gold particles display greater labeling efficiency because of the reduced steric hindrance (Carrassi et al, 1990).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Application of ion etching to immunoscanning electron microscopy

Yahiro

Nagato

2005

Microsc. Res. Tech.

View full text Add to dashboard Cite

A method for achieving both the light and electron microscopic observations of the same immunolabeled semithin section is described. Mild ion etching (IE) was performed on the semithin LR white resin sections of rat pancreas to evaluate conditions for scanning electron microscopic secondary electron image observations. Before immunocytochemical staining, very mild, rapid etching was conducted as follows: ionization voltage 300 V, operating vacuum 35 Pa, and etching time 1 min, employing an ion coater above sections on glass slides. The sections were immunohistochemically stained with anti-insulin and immunogold in association with silver enhancement techniques for light microscopic observation, in which B cells in pancreatic islets were positively stained brown. Subsequently, essential mild IE was performed over the stained section as follows: 350 V, 38 Pa, 29 min. The samples were coated with platinum for scanning electron microscopic secondary electron images, in which the cores of secretory granules of the B cells were positively labeled with gold-silver particles. The present method is suitable for detection of substances involving immunogold labeling. It enables us to obtain high-resolution images at low magnification that can be correlated with light microscopic observations. Middle to high magnifications are applicable for detailed observations with secondary electron imaging scanning electron microscopy.

show abstract

Section: Discussionsupporting

confidence: 87%

Section: Discussionmentioning

confidence: 99%

Application of ion etching to immunoscanning electron microscopy

Yahiro

Nagato

2005

Microsc. Res. Tech.

View full text Add to dashboard Cite

show abstract

“…4,18 These techniques compliment more common methods for mapping proteins such as laser scanning microscopy of fluorescent labeling proteins, 19 which has high specificity but limited spatial resolution, and immunoelectron microscopy, which has high spatial resolution but requires a chemically fixed sample. 20 The X-ray absorption spectra of proteins are quite distinct from that of other biological materials, especially at the C 1s edge. This is the basis for mapping locations and concentrations of proteins relative to other biological materials or carbon-based polymers.…”

Section: Introductionmentioning

confidence: 99%

Using Intrinsic X-ray Absorption Spectral Differences To Identify and Map Peptides and Proteins

et al. 2007

View full text Add to dashboard Cite

The intrinsic variation in the near-edge X-ray absorption fine structure (NEXAFS) spectra of peptides and proteins provide an opportunity to identify and map them in various biological environments, without additional labeling. In principle, with sufficiently accurate spectra, peptides (<50 amino acids) or proteins with unusual sequences (e.g., cysteine-or methionine-rich) should be differentiable from other proteins, since the NEXAFS spectrum of each amino acid is distinct. To evaluate the potential for this approach, we have developed X-SpecSim, a tool for quantitatively predicting the C, N, and O 1s NEXAFS spectra of peptides and proteins from their sequences. Here we present the methodology for predicting such spectra, along with tests of its precision using comparisons to the spectra of various proteins and peptides. The C 1s, N 1s, and O 1s spectra of two novel antimicrobial peptides, Indolicidin (ILPWKWPWWPWRR-NH 2 ) and Sub6 (RWWKIWVIR-WWR-NH 2 ), as well as human serum albumin and fibrinogen are reported and interpreted. The ability to identify, differentiate, and quantitatively map an antimicrobial peptide against a background of protein is demonstrated by a scanning transmission X-ray microscopy study of a mixture of albumin and sub6.

show abstract

“…The IEM technique using colloidal gold particles has been known for some time to be useful and reliable in the localization of various classes of cell antigens (6,22,26,44,(48)(49)(50), although, to our knowledge, it has not been used to detect serum antibodies. The only IEM study performed with human sera as the source of specific antibodies dates back to 1977.…”

Section: Discussionmentioning

confidence: 99%

Immunoelectron microscopy: a new method for detection of insulin antibodies.

Saccomanno

Taccagni

Bosi

et al. 1993

J Histochem Cytochem.

View full text Add to dashboard Cite

The aim of the present study was to set up a sensitive technical alternative to the classical procedures for detection of human insulin antibodies. We developed a method of post-embedding immunoelectron microscopy (IEM) using as the substrate fresh human pancreas, embedded in acrylic resin to maintain its antigenic structure. The antigen was insulin within the mature secretory granule. Serum samples obtained from 10 patients with insulin antibodies detected at various titers by either radio binding assay (RBA) or enzymatic immunoassay (EIA) were incubated for 2 hr at 37 degrees C at dilutions of 1:25, 1:100, 1:400, 1:1600, 1:6400, 1:25,600, and 1:102,400. The electron microscope photographs were analyzed by computerized morphometry and the number of protein A-gold-IgG complexes was calculated per micron2 of insulin granule. IEM results were compared with those obtained with EIA. The specificity of both techniques towards insulin was assessed as the difference between the signals (number of gold particles per micron2 of insulin granule in IEM or optical density > or = 0.193 in EIA) with and without excess insulin. Sensitivity was defined as the detection limit of the assay. In all the 10 sera investigated, IEM was more sensitive, with a 12- to 40-fold lower detection limit than EIA. IEM, with native insulin granules as substrate, is a specific, reproducible, and sensitive method for detection of human serum insulin antibodies. These findings also suggest IEM as a procedure potentially suitable for identifying antigen specificity of autoantibodies circulating at low concentration.

show abstract

Immunolabeling with the Protein A-Gold Technique: An Overview

Cited by 27 publications

References 0 publications

Application of ion etching to immunoscanning electron microscopy

Application of ion etching to immunoscanning electron microscopy

Using Intrinsic X-ray Absorption Spectral Differences To Identify and Map Peptides and Proteins

Immunoelectron microscopy: a new method for detection of insulin antibodies.

Contact Info

Product

Resources

About