An Evaluation of Antigen Retrieval Procedures for Immunoelectron Microscopic Classification of Amyloid Deposits

Röcken, Christoph; Roessner, Albert

doi:10.1177/002215549904701105

Cited by 30 publications

(28 citation statements)

References 26 publications

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“…Moreover, Shi et al (1996) devised the test battery approach, which can efficiently determine the optimum protocols of AR for each antigen by comparing the immunostaining results between different kinds of solutions, temperatures, and pH (O'Leary 2001). As one good example using the test battery approach, it was demonstrated that AR procedures can also be applied to immunoelectron microscopy for amyloid deposits composed of the κ light chain or transthyretin (Rocken and Roessner 1999).…”

Section: Discussionmentioning

confidence: 99%

Enhanced Antigen Retrieval of Amyloid β Immunohistochemistry

Kai

Shin

Ogino

et al. 2012

J Histochem Cytochem.

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Senile plaques, extracellular deposits of amyloid β peptide (Aβ), are one of the pathological hallmarks of Alzheimer disease (AD). As the standard immunohistochemical detection method for Aβ deposits, anti-Aβ immunohistochemistry combined with antigen retrieval (AR) by formic acid (FA) has been generally used. Here, we present a more efficient AR for Aβ antigen. On brain sections of AD and its mouse model, a double combination of either autoclave heating in EDTA buffer or digestion with proteinase K plus FA treatment reinforced Aβ immunoreactivity. A further triple combination of digestion with proteinase K (P), autoclave heating in EDTA buffer (A), and FA treatment (F), when employed in this order, gave a more enhanced immunoreactivity. Our PAF method prominently visualized various forms of Aβ deposits in AD that have not been clearly detected previously and revealed numerous minute-sized plaques both in AD and the mouse model. Quantification of Aβ loads showed that the AR effect by the PAF method was 1.86-fold (in the aged human brain) and 4.64-fold (in the mouse brain) higher than that by the FA method. Thus, the PAF method could have the potential to be the most sensitive tool so far to study Aβ pathology in AD and its mouse model.

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Section: Discussionmentioning

confidence: 99%

Enhanced Antigen Retrieval of Amyloid β Immunohistochemistry

Kai

Shin

Ogino

et al. 2012

J Histochem Cytochem.

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“…For immunohistochemistry and TEM, ultrathin sections were mounted on gold grids and subjected to Ag retrieval with sodium metaperiodate (41,42). For immunostaining, the grids were floated on top of drops of immune reagents displayed on a sheet of parafilm.…”

Section: Fluorescence Confocal and Transmission Electron Microscopymentioning

confidence: 99%

Superantigen- and TLR-Dependent Activation of Tonsillar B Cells after Receptor-Mediated Endocytosis

Jendholm

Mörgelin

Vidakovics

et al. 2009

The Journal of Immunology

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Classical B lymphocyte activation is dependent on BCR cross-linking in combination with physical interaction with Th cells. Other B cell molecules that contribute to the activation are complement, cytokine, and TLRs recognizing specific pathogen-associated molecular patterns. Moraxella (Branhamella) catarrhalis is a common Gram-negative respiratory pathogen that induces proliferation in human IgD-expressing B cells independently of T cell help. The activation is initiated by the B cell superantigen Moraxella IgD-binding protein (MID) through a nonimmune cross-linking of IgD. However, IgD cross-linking alone is not sufficient to induce proliferation. In this study, we characterized the significance of TLRs in superantigen-dependent B cell activation using whole bacteria or rMID in the presence or absence of TLR ligands. IgD cross-linking by MID sensitized B cells obtained from children with tonsillar hyperplasia for mainly TLR9, whereas TLRs 1, 2, 6, and 7 were less important. The Moraxella-induced activation was inhibited when a dominant-negative TLR9 ligand was added. Interestingly, BCR-mediated endocytosis of whole Moraxella and degradation of live bacteria in naive B cells were observed with fluorescence, confocal, and transmission electron microscopy. This unique observation proved the strong intracellular TLR9 response as well as highlighted the Ag-presenting function of B cells. In conclusion, our findings suggest an important role of TLRs in the adaptive immune response and reveal novel insights into the T cell-independent B cell activation induced by bacteria.

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“…Immunoelectron microscopy was performed in triplicate as described previously. 17 The specificity of immunostaining was controlled by omitting the primary antibody. The sections were air-dried and examined using a Zeiss EM900 electron microscope.…”

Section: Electron Microscopymentioning

confidence: 99%

Proteolysis of AA Amyloid Fibril Proteins by Matrix Metalloproteinases-1, -2, and -3

Stix

Kähne

Sletten

et al. 2001

The American Journal of Pathology

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We recently demonstrated the presence of matrix metalloproteinases (MMPs)-1, -2, and -3 in AA amyloid deposits, which lead us to speculate that MMPs may participate in amyloidogenesis by either processing the precursor protein, or by degrading the amyloid deposits. Here we investigated this theory by determining the ability of MMP-1, -2, and -3 to degrade human acute-phase serum amyloid A (SAA) and human AA amyloid fibril proteins (AFPs). The following in vitro degradation experiments were performed: using either recombinant MMP-1, -2, or -3 and SAA as a substrate; using either recombinant MMP-1, -2, or -3 and AFP as a substrate; and using THP-1 cells as the protease source and AFP as the substrate. All three MMPs were able to cleave SAA and AFP within the region spanning residues 51 to 57. The following cleavage sites were identified: at 57 to 58 for MMP-1; at 7 to 8 and 51 to 52 for MMP-2; at 7 to 8, 16 to 17, 23 to 24, 51 to 52, 55 to 56, 56 to 57, and 57 to 58 for MMP-3. Cell culture experiments showed that THP-1 cells were able to degrade AFPs. Degradation was significantly delayed after addition of a general metalloproteinase inhibitor (o-phenanthroline) to dextran sulfate-stimulated cells. This is the first study to show that human SAAs and AFPs are susceptible to proteolytic cleavage by MMPs. Immunocytochemistry and electron microscopy showed that degradation takes place in the pericellular or extracellular compartment. (Am J Pathol 2001, 159:561-570)

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An Evaluation of Antigen Retrieval Procedures for Immunoelectron Microscopic Classification of Amyloid Deposits

Cited by 30 publications

References 26 publications

Enhanced Antigen Retrieval of Amyloid β Immunohistochemistry

Enhanced Antigen Retrieval of Amyloid β Immunohistochemistry

Superantigen- and TLR-Dependent Activation of Tonsillar B Cells after Receptor-Mediated Endocytosis

Proteolysis of AA Amyloid Fibril Proteins by Matrix Metalloproteinases-1, -2, and -3

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