scholarly journals ULTRASTRUCTURAL LOCALIZATION OF ANTIBODY IN DIFFERENTIATING PLASMA CELLS

1968 ◽  
Vol 127 (1) ◽  
pp. 109-118 ◽  
Author(s):  
Elizabeth H. Leduc ◽  
Stratis Avrameas ◽  
Michel Bouteille
Keyword(s):  
Electron Microscopy ◽  
Golgi Apparatus ◽  
Horseradish Peroxidase ◽  
Peroxidase Activity ◽  
Plasma Cells ◽  
Ultrastructural Localization ◽  
Fixed Tissue ◽  
Plasmacytic Differentiation ◽  
Perinuclear Space ◽  
Large Spherical

Antibody was localized by electron microscopy within differentiating and mature plasma cells of the spleens of hyperimmunized rabbits. Horseradish peroxidase was used as antigen. Intracellular antibody to peroxidase was revealed in glutaraldehyde-fixed tissue by coupling it with its antigen and then revealing the sites of peroxidase activity cytochemically. Antibody first appears in the perinuclear space of hemocytoblasts where it persists through differentiation into immature plasma cells, but it disappears from this site in mature plasma cells. Concomitant with the development of the ergastoplasm, antibody accumulates in many but not all of its cisternae. Antibody is present in the lamellar portion of the Golgi apparatus in all phases of plasmacytic differentiation. Mature plasma cells exhibit two types of antibody distribution, a concentration into large spherical intracisternal granules or an overflowing into all parts of the cytoplasm.

Comparative strengths and weaknesses of peroxidase and colloidal gold in pre- and post-embedding immunolabeling techniques

1987 ◽  
Vol 45 ◽  
pp. 1002-1005
Author(s):  
D. R. Abrahamson ◽  
P. L. St.John ◽  
E. W. Perry
Keyword(s):  
Electron Microscopy ◽  
Horseradish Peroxidase ◽  
Light Microscopy ◽  
Colloidal Gold ◽  
Light Microscope ◽  
Ultrastructural Localization ◽  
The Other ◽  
Quantitative Studies ◽  
Dense Suspension ◽  
Antigen Localization

Antibodies coupled to tracers for electron microscopy have been instrumental in the ultrastructural localization of antigens within cells and tissues. Among the most popular tracers are horseradish peroxidase (HRP), an enzyme that yields an osmiophilic reaction product, and colloidal gold, an electron dense suspension of particles. Some advantages of IgG-HRP conjugates are that they are readily synthesized, relatively small, and the immunolabeling obtained in a given experiment can be evaluated in the light microscope. In contrast, colloidal gold conjugates are available in different size ranges and multiple labeling as well as quantitative studies can therefore be undertaken through particle counting. On the other hand, gold conjugates are generally larger than those of HRP but usually can not be visualized with light microscopy. Concern has been raised, however, that HRP reaction product, which is exquisitely sensitive when generated properly, may in some cases distribute to sites distant from the original binding of the conjugate and therefore result in spurious antigen localization.

scholarly journals The ultrastructural localization of immunoglobulins in human b cells of immunoproliferative diseases

Blood ◽  
1982 ◽  
Vol 59 (6) ◽  
pp. 1132-1140 ◽  
Author(s):  
MF Gourdin ◽  
JP Farcet ◽  
F Reyes
Keyword(s):  
Endoplasmic Reticulum ◽  
B Cells ◽  
Plasma Cells ◽  
Simultaneous Detection ◽  
Ultrastructural Localization ◽  
Lymphocytic Leukemia ◽  
Cellular Distribution ◽  
Complex Plasma ◽  
Perinuclear Space ◽  
Human B Cells

Abstract The cellular distribution of immunoglobulins in human malignant and normal B cells was investigated by immunoelectron microscopy by direct incubation of fixed cells with electron microscopy by direct incubation of fixed cells with peroxidase-coupled antibody. These conjugates penetrated into the cell, resulting in the simultaneous detection of surface and cytoplasmic immunoglobulins. The latter were seen as specific intracisternal staining of the perinuclear space and endoplasmic reticulum and occasionally of the Golgi complex. Plasma cells were frequently characterized by a heterogeneity of reactivity of the endoplasmic reticulum. Minute amounts of cytoplasmic immunoglobulin were demonstrated in cells without developed secretory organelles, such as lymphoma cells and lymphocytes from chronic lymphocytic leukemia (CLL). The method allowed us to define several subsets of cells according to the expression of surface and cytoplasmic immunoglobulins and thus to determine the stage of maturation of cells involved in monoclonal proliferation.

scholarly journals Immune complex receptors on cell surface. I. Ultrastructural demonstration of macrophages.

1976 ◽  
Vol 24 (8) ◽  
pp. 948-955 ◽  
Author(s):  
P E McKeever ◽  
A J Garvin ◽  
S S Spicer
Keyword(s):  
Electron Microscopy ◽  
Horseradish Peroxidase ◽  
Cell Surface ◽  
Immune Complex ◽  
Immune Complexes ◽  
External Surface ◽  
Binding Capacity ◽  
Ultrastructural Localization ◽  
Soluble Complex ◽  
Receptor Sites

A method is described for ultrastructural localization of immune complex receptors on the surface of viable peritoneal exudate cells. The technique entails incubation with a soluble complex of horseradish peroxidase (HRP) and specific antibody to HRP at 4 degrees C followed by exposure to diaminobenzidine and processing for electron microscopy. The bound immune complexes were evident as focal deposits of HRP reaction product, adhering closely to the external surface of macrophages with an uninterrupted periodicity varying between 30 and 120 nm. Following incubation with an insoluble immune complex containing a higher proportion of antibody, receptor sites stained frequently, but large aggregates adhered to the cells. Rinsing cells after staining with soluble complexes partially displaced the bound immune complexes. Fixation prior to exposure to immune complexes largely eliminated the binding capacity of the immune complex receptors.

scholarly journals The ultrastructural localization of immunoglobulins in human b cells of immunoproliferative diseases

Blood ◽  
1982 ◽  
Vol 59 (6) ◽  
pp. 1132-1140
Author(s):  
MF Gourdin ◽  
JP Farcet ◽  
F Reyes
Keyword(s):  
Endoplasmic Reticulum ◽  
B Cells ◽  
Plasma Cells ◽  
Simultaneous Detection ◽  
Ultrastructural Localization ◽  
Lymphocytic Leukemia ◽  
Cellular Distribution ◽  
Complex Plasma ◽  
Perinuclear Space ◽  
Human B Cells

The cellular distribution of immunoglobulins in human malignant and normal B cells was investigated by immunoelectron microscopy by direct incubation of fixed cells with electron microscopy by direct incubation of fixed cells with peroxidase-coupled antibody. These conjugates penetrated into the cell, resulting in the simultaneous detection of surface and cytoplasmic immunoglobulins. The latter were seen as specific intracisternal staining of the perinuclear space and endoplasmic reticulum and occasionally of the Golgi complex. Plasma cells were frequently characterized by a heterogeneity of reactivity of the endoplasmic reticulum. Minute amounts of cytoplasmic immunoglobulin were demonstrated in cells without developed secretory organelles, such as lymphoma cells and lymphocytes from chronic lymphocytic leukemia (CLL). The method allowed us to define several subsets of cells according to the expression of surface and cytoplasmic immunoglobulins and thus to determine the stage of maturation of cells involved in monoclonal proliferation.

Localization of Acetylcholine Receptors with Peroxidase-Labeled α-Bungarotoxin

1976 ◽  
Vol 34 ◽  
pp. 8-9 ◽  
Author(s):  
Thomas L. Lentz ◽  
Janice Chester ◽  
Jean Rosenthal
Keyword(s):  
Electron Microscopy ◽  
Horseradish Peroxidase ◽  
Peroxidase Activity ◽  
Acetylcholine Receptors ◽  
Osmium Tetroxide ◽  
Physiological Activity ◽  
Cytochemical Localization ◽  
Snake Neurotoxin ◽  
Frog Sartorius ◽  
Newt Limb

A procedure has been developed for the cytochemical localization of acetylcholine (ACh) receptors by means of the snake neurotoxin α-bungarotoxin (α-Btx) labeled directly with horseradish peroxidase (HRP). Conjugation of HRP to α-Btx was carried out according to the method of Nakane and Kawaoi (1). A fraction of the conjugate with a MW of ∿48,000 was separated from free peroxidase and heavier MW fractions by column chromatography. This fraction corresponds to a conjugate in which one molecule of HRP is coupled to one of α-Btx. It was found to retain physiological activity since it produced a postsynaptic blockade at frog sartorius neuromuscular junction similar to that obtained with native α-Btx. For localization of receptors, mouse diaphragm, frog sartorius, or newt limb muscles were incubated in the conjugate for from one half to two hours, fixed in glutaraldehyde, reacted with H2O2 and diaminobenzidine for peroxidase activity, fixed in osmium tetroxide, and processed for electron microscopy.

scholarly journals ULTRASTRUCTURAL LOCALIZATION OF INTRACELLULAR IMMUNE GLOBULINS IN PLASMA CELLS AND LYMPHOBLASTS BY ENZYME-LABELED ANTIBODIES

1969 ◽  
Vol 17 (4) ◽  
pp. 211-224 ◽  
Author(s):  
ELIZABETH H. LEDUC ◽  
GEOFFREY B. SCOTT ◽  
STRATIS AVRAMEAS
Keyword(s):  
Immune Response ◽  
Plasma Cells ◽  
Ultrastructural Localization ◽  
Frozen Sections ◽  
Cytoplasmic Staining ◽  
Specific Antibodies ◽  
Perinuclear Space ◽  
Cacodylate Buffer ◽  
Immune Globulins ◽  
Ultrathin Frozen Sections

Techniques for localization of immune globulins by specific antibodies labeled with peroxidase or alkaline phosphatase were applied to spleens of rabbits hyperimmunized against various antigens. Best results were obtained with tissue blocks or thick sections after fixation in 1% formaldehyde (freshly prepared from paraformaldehyde) in cacodylate buffer with sucrose. The edges of blocks gave adequate penetration, comparable to thick sections. Ultrathin frozen sections which were reacted directly with labeled conjugate were less successful and glycol methacrylate sections failed to stain. Results were 3-fold: (1) immune globulins were demonstrated in ergastoplasmic cisternae, perinuclear space and Golgi apparatus of plasmocytes; (2) associated ribosomal staining varied somewhat with fixation and osmolarity of fixative; (3) lymphoblasts were shown to contain antibody. Generalized cytoplasmic staining was observed in the lymphoblasts, with no localization in the ergastoplasm and with no staining of control preparations. The significance of ribosomal and lymphoblast staining is discussed in relation to cell differentiation and the development of the immune response.

scholarly journals LOCALIZATION OF ANTIBODIES BY ELECTRON MICROSCOPY IN DEVELOPING ANTIBODY-PRODUCING CELLS

1965 ◽  
Vol 26 (3) ◽  
pp. 759-778 ◽  
Author(s):  
S. de Petris ◽  
Gioanna Karlsbad
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Plasma Cells ◽  
Early Stage ◽  
Cell Types ◽  
Antibody Synthesis ◽  
Booster Injection ◽  
Perinuclear Space ◽  
Lymph Node Cells ◽  
Antigen Antibody

The development of antibody-producing cells in the early stages of the secondary or hyperimmune response has been studied with the electron microscope in lymph nodes of adult chinchilla rabbits immunized with ferritin or apoferritin. The intracellular distribution of antiferritin antibodies was determined in the lymph node cells at 1 to 5 days after a booster injection, employing the labelling technique previously used by the authors (12) to demonstrate the localization of antibodies in mature plasma cells. Antibodies were first detected at 48 hours in blasts; i.e., cells which have a poorly developed endoplasmic reticulum and a cytoplasm filled with many ribosomes grouped in clusters. The label was subsequently found in forms intermediate between blasts and plasma cells (plasmoblasts, immature plasma cells), in which the endoplasmic reticulum appeared progressively more developed. Antiferritin antibodies were also found in cells in mitosis. In all the above cell types, antigen-antibody precipitates were consistently found in the perinuclear space and in the cisternae of the granular endoplasmic reticulum, from an early stage in the development of the latter. Evidence was also obtained for the presence of antibody in the Golgi area. The results are discussed in relation to the possible cellular sites of antibody synthesis.

scholarly journals Immunocytochemical demonstration of intracytoplasmic alkaline phosphatase in HeLa TCRC-1 cells.

1981 ◽  
Vol 29 (9) ◽  
pp. 1080-1087 ◽  
Author(s):  
S Tokumitsu ◽  
K Tokumitsu ◽  
W H Fishman
Keyword(s):  
Alkaline Phosphatase ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Distribution Patterns ◽  
Ultrastructural Localization ◽  
Surface Plasma ◽  
Membrane Glycoproteins ◽  
Perinuclear Space ◽  
Ectopic Gene Expression

The ultrastructural localization of alkaline phosphatase has been examined in cells of a HeLa subline (TCRC-1) that are monophenotypic for Regan isoenzyme expression. Enzyme activity was demonstrated at the cell surface plasma membrane and in certain lysosomes as revealed by the lead citrate method. The regular direct immunoperoxidase procedure utilizing antibodies in IgG or Fab' form showed the same distribution patterns of alkaline phosphatase. However, when the cell surface antigen was blocked in advance with specific unlabeled antibodies and direct immunocytochemistry performed in the presence of saponin, intracellular alkaline phosphatase antigen was observed in the perinuclear space, endoplasmic reticulum, and Golgi apparatus. The results appeared to be concordant with the current concept that membrane glycoproteins are formed in the endoplasmic reticulum, modified in the Golgi apparatus and then transported to the cell surface. Intracellular alkaline phosphatase was observed predominantly in some cell populations especially mitotic cells, suggesting that the enzyme protein was synthesized in and around the mitotic phase. Accordingly, this technique of differential membrane immunocytochemistry appears to provide an opportunity to follow ectopic gene expression as a function of cell cycle and enzyme induction.

scholarly journals CYTOCHEMICAL DETECTION OF SITES OF ANTIBODY TO HORSERADISH PEROXIDASE IN SPLEEN AND LYMPH NODES

1968 ◽  
Vol 16 (4) ◽  
pp. 237-248 ◽  
Author(s):  
WERNER STRAUS
Keyword(s):  
Horseradish Peroxidase ◽  
Lymph Nodes ◽  
Plasma Cells ◽  
Specific Binding ◽  
Repeated Injection ◽  
Similar Reaction ◽  
Fixed Tissue ◽  
Tissue Sections ◽  
Spleen And Lymph Nodes

After repeated injection of small amounts of horseradish peroxidase into the perivertebral muscle and footpads of rabbits, certain sites in spleen and popliteal lymph nodes showed intense adsorption of antigen (horseradish peroxidase) during treatment of fixed tissue sections in vitro. The reaction occurred in certain cells, probably plasma cells, and was also positive in the reticulum extending between lymphocytes. These sites were considered to contain antibodies against horseradish peroxidase, since sections from the same block, not treated with antigen in vitro, and corresponding sections from nonimmunized rabbits did not give a similar reaction. The blood sera of rabbits which showed specific binding of antigen in the spleen caused approximately 60% inhibition of horseradish peroxidase activity, but no inhibition was caused by the blood sera of nonimmunized control animals.

scholarly journals LOCALIZATION OF ANTIBODIES IN PLASMA CELLS BY ELECTRON MICROSCOPY

1963 ◽  
Vol 117 (5) ◽  
pp. 849-862 ◽  
Author(s):  
Stefanello de Petris ◽  
Gioanna Karlsbad ◽  
Benvenuto Pernis
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Cell Membrane ◽  
Plasma Cells ◽  
Antibody Concentration ◽  
Outer Cell ◽  
Perinuclear Space ◽  
Outer Cell Membrane ◽  
Antigen Antibody ◽  
Direct Counts

The localization of antibody in the interior of plasma cells of lymph nodes of rabbits hyperimmunized with ferritin was studied by electron microscopy. The cells were incubated with the antigen (ferritin) which was allowed to penetrate into the cells by suitable methods. Antigen-antibody precipitates were localized in the cisternae of the endoplasmic reticulum and in the perinuclear space. No evident association was found between ferritin and ribosomes or ferritin and outer cell membrane. Cells from control animals immunized with an unrelated antigen, incubated with ferritin, exhibited no labeling. From direct counts of ferritin molecules in plasma cells, a lower limit was evaluated for the antibody concentration in the endoplasmic reticulum (12.2 mg/cm3) and for the total antibody content of a plasma cell (7.0 x 10–3 gm).

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