Localization of Antibodies in Plasma Cells by Electron Microscopy

Petris, S. de; Karlsbad, Gioanna; Pernis, Benvenuto

doi:10.1084/jem.117.5.849

Cited by 92 publications

(29 citation statements)

References 22 publications

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“…For electron microscopy, some thin tissue slices were fixed in 1 per cent osmium tetroxide buffered according to Palade (36) or Millonig (28), dehydrated in a graded series of alcohols and embedded in Araldite according to the standard techniques (27). Most of the lymph node tissue was used instead tot labelling the intracellular antibodies with FT. A detailed description of the technique employed is given in our previous paper (12). Most of the present experiments were carried out on isolated lymph node cells.…”

Section: Methodsmentioning

confidence: 99%

“…In some experiments the labelling technique was applied to whole lymph node sections as well as to isolated cells (12) : in this case, slices of lymph node were fixed for 1 hour with 4 per cent buffered formaldehyde, infiltrated with 10% dimethyl sulfoxide (for about 1 hour), and slowly frozen in a "cold box." Sections 15 to 20 # thick were cut in a cryostat, incubated with FT, washed, and prepared for electron microscopy as described previously (12).…”

Section: Methodsmentioning

confidence: 99%

“…In a previous paper (12) de Petris, Karlsbad, and Pernis described the subcellular distribution of antiferritin antibodies in the typical plasma cells present in lymph nodes of hyperimmunized rabbits on the 4th and 5th day after a booster injection of antigen. The antibodies (marked with ferritin) appeared to be localized in the perinuclear space and cisternae of the endoplasmic reticulum, and possibly also in the Golgi region.…”

mentioning

confidence: 99%

“…The technique employed for labelling the antibodies was essentially the same as that used in the previous investigation (12): it consisted in incubating ferritin with lymph node cells of animals immunized with either ferritin or apoferritin, after suitable treatment aimed at allowing the penetration of the marker (ferritin) into the ceils. The localization of antibodies was deduced from the distribution of ferritin-antiferritin microprecipitates.…”

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confidence: 99%

“…Preliminary communications on this subject were presented at the Padua and Prague meetings of electron microscopy (10).…”

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confidence: 99%

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Localization of Antibodies by Electron Microscopy in Developing Antibody-Producing Cells

Petris

Karlsbad

1965

The Journal of Cell Biology

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The development of antibody-producing cells in thc early stagcs of the sccondary or hypcrimmune response has becn studied with the electron microscope in lymph nodes of adult chinchilla rabbits immunized with fcrritin or apofcrritin. The intraccllular distribution of antifcrritin antibodies was determined in the lymph node cells at 1 to 5 days after a booster injcction, employing the labelling technique prcviously used by the authors (12) to dcmonstratc the localization of antibodies in mature plasma cells. Antibodies were first detected at 48 hours in blasts; i.e., cells which have a poorly dcveloped endoplasmic reticulum and a cytoplasm filled with many ribosomes grouped in clusters. The label was subscquently found in forms intermediate bctwccn blasts and plasma cells (plasmoblasts, immature plasma cells), in which the cndoplasmic reticulum appeared progressivcly more dcvclopcd. Antifcrritin antibodies wcrc also found in cells in mitosis. In all the above ccll types, antigcnantibody precipitates were consistently found in the perinuclear space and in the cisternae of the granular endoplasmic rcticulum, from an early stage in the development of thc latter. Evidcncc was also obtained for thc presence of antibody in the Golgi area. The results arc discussed in relation to the possible cellular sites of antibody synthesis.In a previous paper (12) de Petris, Karlsbad, and Pernis described the subcellular distribution of antiferritin antibodies in the typical plasma cells present in lymph nodes of hyperimmunized rabbits on the 4th and 5th day after a booster injection of antigen. The antibodies (marked with ferritin) appeared to be localized in the perinuclear space and cisternae of the endoplasmic reticulum, and possibly also in the Golgi region.We have now extended this research to the early stages of the immune response, namely to the study of the cells present in the lymph nodes at 24 to 72 hours after restimulation. These early stages are characterized by active cellular division, accompanied by a series of cellular transformations entailing marked ultrastructural changes. In an effort to throw light on the cellular aspects of antibody synthesis, we have tried to correlate the beginning and further development of the antibody-synthesizing activity, as deduced from the initial and subsequent intracellular distribution of labelled antibodies, with the morphological changes which occur in the antibody-producing cells and eventually lead to their full differentiation into plasma cells.The technique employed for labelling the antibodies was essentially the same as that used in the previous investigation (12): it consisted in incubating ferritin with lymph node cells of animals immunized with either ferritin or apoferritin, after suitable treatment aimed at allowing the penetration of the marker (ferritin) into the ceils. The localization of antibodies was deduced from the distribution of ferritin-antiferritin microprecipitates.Preliminary communications on this subject 759

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

“…Preliminary communications on this subject were presented at the Padua and Prague meetings of electron microscopy (10).…”

mentioning

confidence: 99%

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Localization of Antibodies by Electron Microscopy in Developing Antibody-Producing Cells

Petris

Karlsbad

1965

The Journal of Cell Biology

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Ultrastructure of the myeloma cell

Maldonado

Brown

Bayrd

et al. 1966

Cancer

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An evaluation of the ultrastructure of the myeloma cell in 19 cases confirms its marked pleomorphism. It ranges from a cell indistinguishable from the normal plasmacyte or its precursors to a cell with clearly abnormal configuration. Abnormal features included abnormalities in size and shape of the cell, nucleus, nucleolus and mitochondria; nucleocytoplasmic asynchronism; disorganization, atypical location and hypertrophy of the Golgi apparatus; and the presence of multiple centrioles. Six types of arrangement of the endoplasmic reticulum were differentiated. In certain cases there were cells with seemingly intermediate features between the lymphocyte or the reticulum cell and the plasma‐myeloma cell. Intranuclear and different types of cytoplasmic dense (osmiophilic) bodies were observed. These bodies likely represent condensed protein and some may be lysosomes. Some morphologic features suggest that the Golgi apparatus plays a role in the condensing process. The myeloma cell releases its secretion mainly by clasmatosis or cytoplasmic fragmentation.

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Immunohistochemical identification of immunoglobulin A on ultrathin tissue sections

1980

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Antigenic determinants of the heavy chain of immunoglobulin A (IgA) were identified within immunocytes by immunohistochemical techniques applied t o ultrathin sections of rat ileal mucosa embedded in epoxy resin. Successful localization of IgA depended upon the utilization of chemical fixatives containing paraformaldehyde or physical fixation employing a freeze-drying technique. Partial removal of the epoxy resin from the ultrathin tissue section with metallic alkoxide solutions or pretreatment with H 2 0 2 was essential prior t o the sequential application of the immunological reagents. IgA was associated with the rough endoplasmic reticulum (RER) and nuclear regions of immunocytes. These results are similar to those of other investigators employing immunochemical localization before embedment.Ultrastructural identification of immunoglobulin antigenic determinants by the immunoferritin-(dePetris e t al., '63) and peroxidase-conjugated antibody (Avrameas, '7 1); Rrown et al., '76; Leduc e t al., '69) immunohistochemical methods have previously required the use of these techniques prior to the embedding process. Recently, the successful light microscopic identification of light and heavy chains of IgA on sections of rat ileal mucosa embedded in epoxy resin was reported (Rodning e t al., '79). In the present investigation we have demonstrated that IgA antigens can he identified by the unlabeled antibody enzyme method on ultrathin sections of tissue embedded in epoxy resin. Before immunocytochemical staining, sections were etched for 20-30 minutes with 10% H 2 0 2 , or for 1-5 minutes with a metallic alkoxide solution consisting of absolute methanol or ethanol saturated with NaOH (Lane and Europa, '65). The ultrathin tissue sections were then stained immunohistochemically for rat IgA by a modification of the unlabeled antibody enzyme method (Erlandsen et al., '76; Rodning et al., '79). The specificity of immunological reactions was determined by the following controls: 1) by employing normal rabbit serum in place of the specific primary antiserum; 2) by absorbing the specific primary antiserum with purified antigen; and 3) by substituting normal sheep serum for sheet anti-rabbit IgG. MATERIALS AND METHODSRESULTS Large numbers of immunocytes are routinely found within the lamina propria of the rat ileal mucosa (Fig. 1). The successful immunocytochemical localization of IgA in immunocytes at the ultrastructural level is shown in Figures 2 and 3 , but with an overall compromise in the preservation of cytoplasmic and extracellular detail. In immunocytes from tissue fixed in paraformaldehyde (Fig. 2), immunocytochemical staining for IgA was detected within structures resembling parallel arrays of RER and in the nuclear region corresponding to the nuclear cisternae and heterochromatin. As shown in Figure 3, physical fixation by freeze drying also permitted successful staining of IgA within the RER and perinuclear area; however, the preservation of ultrastructural detail was severely compromised by artifacts rela...

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Localization of Antibodies in Plasma Cells by Electron Microscopy

Cited by 92 publications

References 22 publications

Localization of Antibodies by Electron Microscopy in Developing Antibody-Producing Cells

Localization of Antibodies by Electron Microscopy in Developing Antibody-Producing Cells

Ultrastructure of the myeloma cell

Immunohistochemical identification of immunoglobulin A on ultrathin tissue sections

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