Double labelling of cell surface antigens with colloidal gold markers

Harven, Étienne de; Soligo, Davide; Christensen, Hilary

doi:10.1007/bf01962875

Cited by 14 publications

(2 citation statements)

References 22 publications

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“…Admittedly, imaging of surface morphology alone by routine SEM is far from an infallible way to distinguish between T-and B-derived lymphocytes. Expectedly, double immuno-labelling techniques (de Harven et al, 1990) will reveal more convincingly the B-or T-cell derivation of the rare B-ly7 + lymphocytes. Labelling with gold markers of distinct sizes (15 and 30 nm, for example, or 5 and 20 nm if a very high resolution SEM is available) should be performed for B-ly71CD3 and for B-ly7ICD20.…”

Section: Lymphocytesmentioning

confidence: 99%

Immunogold labelling of leukemic hairy cells with the B‐ly7 monoclonal antibody: An SEM and TEM study

Harven

Christensen

Poppema

et al. 1994

Microscopy Res & Technique

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Two cases of hairy cell leukemia have been studied by immuno-TEM and immuno-SEM after immunogold labelling of the cell surface antigen recognized by the B-ly7 monoclonal antibody. Most hairy cells appeared significantly labeled, although the density of the expression of the antigen, as demonstrated by immunogold labelling, seems variable from cell to cell. Moreover, some cells with the morphology of hairy cells and which could not be identified as monocytes were not labeled. Labelling for the antigen identified by the B-ly7 mAb does not seem to correlate with the presence of ribosome lamellae complexes which were present only in one of the two cases studied. Rare lymphocytes of unidentified lineage were labeled. Monocytes were significantly absent from the samples of peripheral blood of the two patients studied. In one normal control sample, monocytes were observed unlabelled. The results are discussed in reference to the pathogenesis of hairy cell leukemia, its surprisingly low mitotic rate, and its distinct response to chemotherapy.

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Section: Lymphocytesmentioning

confidence: 99%

Immunogold labelling of leukemic hairy cells with the B‐ly7 monoclonal antibody: An SEM and TEM study

Harven

Christensen

Poppema

et al. 1994

Microscopy Res & Technique

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“…BE1 is advantageous over the secondary electron imaging (SEI) in that the metal coating, needed for SEI, does not obscure the label either as a coating or as a competing element with high atomic number. The high backscatter electron (BE) yield of the gold particles thus offers high contrast, and smaller probes and a wider range of probe sizes can be applied, being ideal for double labeling (de Harven and Soligo, 1989;de Harven et al, 1990;Namork and Heier, 1989;Namork et al, 1987Namork et al, ,1988Namork, 1991;Pawley and Albrecht, 1988;Soligo et al, 1986). Indirect immunolabeling using primary antibodies of different immunoglobulin classes, species, or different ligands was always used in these studies to avoid cross reactivity.…”

Section: Introductionmentioning

confidence: 99%

Backscatter electron imaging of double immunogold labeled erythrocytes using two primary monoclonal IgM antibodies

Heier

1994

Microscopy Res & Technique

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The majority of mouse monoclonal antibodies reacting with blood group epitopes on erythrocytes are of the IgM class, have equal light chain type, and are available as culture supernatants only. To study the interrelationship of the blood group antigens, a method is presented which allows double labeling applying two unconjugated monoclonal antibodies of the same class and species. The method comprises two indirect, sequential labelings using mouse IgM anti-A and anti-H as primary antibodies and two goat anti-mouse IgM conjugated to 30 and 20 nm colloidal gold particles as secondary antibodies. After labeling for the first antigen, free binding sites on the primary antibody are blocked by incubation with an unconjugated goat anti-mouse antibody. The free anti-species on the secondary antibody, conjugated to 30 nm gold particles, are inactivated by silver enhancement. The silver enhancement also enlarges the gold particle for optimal discrimination between the two particle sizes, which are chosen accordingly. Semiquantitations of double labeled cells from subgroup A2 and A3 were found to be in good agreement with the counts of the corresponding single labelings as well as between experiments, irrespective of which of the two antibodies was applied in the first labeling sequence. The results were in accordance with a reciprocal but nonlinear relationship between the A and H antigens and suggest different affinities of the two antibodies for the epitopes in the subgroups investigated, indicating different biochemistry of the antigen determinants.

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Immunogold-Silver Staining (IGSS) for Immunoelectron Microscopy and in Multiple Detection Affinity Cytochemistry

Krenács

1994

Modern Methods in Analytical Morphology

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Double labelling of cell surface antigens with colloidal gold markers

Cited by 14 publications

References 22 publications

Immunogold labelling of leukemic hairy cells with the B‐ly7 monoclonal antibody: An SEM and TEM study

Immunogold labelling of leukemic hairy cells with the B‐ly7 monoclonal antibody: An SEM and TEM study

Backscatter electron imaging of double immunogold labeled erythrocytes using two primary monoclonal IgM antibodies

Immunogold-Silver Staining (IGSS) for Immunoelectron Microscopy and in Multiple Detection Affinity Cytochemistry

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