Ultrastructural immunocytochemical localization of myosin in cultured fibroblastic cells.

Nonmuscle myosin in the cytoplasm of cultured fibroblastic cells has been localized using light and electron microscopic immunocytochemistry. Antibodies to purified fibroblast myosin were produced in goat and rabbit and purified by affinity chromatography. Light microscopic immunofluorescence localization showed patterns similar to those previously published. Electron microscopic localization using the ethyldimethyl aminopropyl carbodiimide-glutaraldehyde-saponin (EGS) fixation-permeabilization procedure and the ferritin bridge localization method produced quantifiable localization in intracellular sites with well-preserved ultrastructural morphology. Myosin was found to be a major component of the cytosol. It was distributed diffusely with no preferential localization on membranous organelles. Myosin was found to be slightly concentrated on the surface of microfilament-containing structures, including the subplasmalemmal microfilament mat and stress fibers, occasionally with an interrupted periodicity. However, no myosin was found in surface ruffles or microvilli. Morphometric quantitation showed that the majority of the cell's myosin was in the cytosol. This location is compatible with myosin being a component of the microtrabecular lattice of the cytoplasmic ground substance. The concentration of myosin in association with microfilaments was only twice that of the cytosol. This interpretation must be somewhat tempered by the possibility that some myosin bound to tightly packed actin may be inaccessible. The significance of this distribution of myosin in cell function is discussed.

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Correlative light and electron microscopic immunocytochemistry on the same section with colloidal gold.

Journal of Histochemistry & Cytochemistry ◽

10.1177/35.4.3546488 ◽

1987 ◽

Vol 35 (4) ◽

pp. 419-425 ◽

Cited By ~ 16

Author(s):

H Mar ◽

T Tsukada ◽

A M Gown ◽

T N Wight ◽

D G Baskin

Keyword(s):

Colloidal Gold ◽

High Specificity ◽

Ultrastructural Localization ◽

Electron Microscopic ◽

Electron Microscopic Immunocytochemistry ◽

Antigenic Sites ◽

Embedding Technique ◽

Positive Immunostaining ◽

Arterial Smooth Muscle Cells ◽

Electron Microscopic Localization

Ultrastructural localization of growth hormone in rat anterior pituitary and of muscle-specific actin in rabbit arterial smooth muscle cells was accomplished with a post-embedment procedure using colloidal gold. Plastic sections (2 microns) were mounted on slides, deplasticized, immunostained with immunoglobulin-colloidal gold particles, re-embedded in Epon, and sectioned for electron microscopy. This procedure enabled light and electron microscopic localization of these intracellular antigens on the same section. Positive immunostaining was demonstrated with this procedure with a muscle-specific actin antibody which previously failed to localize antigenic sites by EM. The procedure described yielded staining of high specificity, with minimal background and well-preserved ultrastructure. This re-embedding technique is useful in situations where problems with post-embedding EM immunostaining exist and where correlative LM and EM immunostaining is essential.

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Observations préliminaires sur l'organisation du cytosquelette des cellules végétales. Mise en évidence d'un “réseau microtrabéculaire”

Canadian Journal of Botany ◽

10.1139/b83-160 ◽

1983 ◽

Vol 61 (5) ◽

pp. 1467-1475 ◽

Cited By ~ 3

Author(s):

Jean-Pierre Arsanto

Keyword(s):

Actin Filaments ◽

Close Relationships ◽

Plant Cells ◽

Ground Substance ◽

Animal Cells ◽

Electron Microscopic ◽

Parenchyma Cells ◽

Vascular Parenchyma ◽

Cytoplasmic Ground Substance ◽

Cellular Components

Electron microscopic observations of particularly favourable sections of pericycle and vascular parenchyma cells fixed with glutaraldéhyde have revealed the existence of a network of exceedingly fine filaments in the cytoplasmic ground substance of plant cells. This highly intricated structure to which polysomes seem to be attached interconnects the cytoskeletal fibers and the various other cellular components. It corresponds evidently to the "microtrabecular lattice" described in animal cells. Moreover, this report points out that microfilaments, which are 6- to 8-nm putative actin filaments, contract with microtubules or sheets of agranular reticulum, to form privileged close relationships whose functional significance is discussed.

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Organisation in the Structure of the Cytoplasmic Ground Substance

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100070357 ◽

1978 ◽

Vol 36 (3) ◽

pp. 627-639

Author(s):

K.R. Porter

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Random Distribution ◽

Ground Substance ◽

The Matrix ◽

Cell Processes ◽

Cytoplasmic Ground Substance

Most types of cells are known from their structure and overall form to possess a characteristic organization. In some instances this is evident in the non-random disposition of organelles and such system subunits as cisternae of the endoplasmic reticulum or the Golgi complex. In others it appears in the distribution and orientation of cytoplasmic fibrils. And in yet others the organization finds expression in the non-random distribution and orientation of microtubules, especially as found in highly anisometric cells and cell processes. The impression is unavoidable that in none of these cases is the organization achieved without the involvement of the cytoplasmic ground substance (CGS) or matrix. This impression is based on the fact that a matrix is present and that in all instances these formed structures, whether membranelimited or filamentous, are suspended in it. In some well-known instances, as in arrays of microtubules which make up axonemes and axostyles, the matrix resolves itself into bridges (and spokes) between the microtubules, bridges which are in some cases very regularly disposed and uniform in size (Mcintosh, 1973; Bloodgood and Miller, 1974; Warner and Satir, 1974).

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Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060696 ◽

1967 ◽

Vol 25 ◽

pp. 136-137

Author(s):

J. P. Petrali ◽

E. J. Donati ◽

L. A. Sternberger

Keyword(s):

Endoplasmic Reticulum ◽

Hela Cells ◽

Mammalian Cells ◽

Specific Antiserum ◽

Electron Microscopic ◽

E Coli ◽

Cytoplasmic Membranes ◽

Viral Progeny ◽

Ultrathin Sections ◽

Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

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Artifact-free electron microscopic immunocytochemistry on rat brain tissue

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100085666 ◽

1991 ◽

Vol 49 ◽

pp. 272-273

Author(s):

Veronika Burmeister ◽

N. Ludvig ◽

P.C. Jobe

Keyword(s):

Microscopic Examination ◽

Free Electron ◽

Electron Microscopic Examination ◽

Ultrastructural Localization ◽

Rabbit Serum ◽

Electron Microscopic ◽

Electron Microscopic Immunocytochemistry ◽

Phosphate Buffered Saline ◽

Rat Brain Tissue ◽

Immune Rabbit

Electron microscopic immunocytochemistry provides an important tool to determine the ultrastructural distribution of various molecules in both normal and pathologic tissues. However, the specific immunostaining may be obscured by artifactual immunoreaction product, misleading the investigator. Previous observations show that shortening the incubation period with the primary antibody from the generally used 12-24 hours to 1 hour substantially reduces the artifactual immunostaining. We now extend this finding by the demonstration of artifact-free ultrastructural localization of the Ca2/calmodulindependent cyclic nucleotide phosphodiesterase (CaM-dependent PDE) immunoreactivity in brain.Anesthetized rats were perfused transcardially with phosphate-buffered saline followed by a fixative containing paraformaldehyde (4%) and glutaraldehyde (0.25%) in PBS. The brains were removed, and 40μm sections were cut with a vibratome. The sections were processed for immunocytochemistry as described by Ludvig et al. Both non-immune rabbit serum and specific CaM-dependent PDE antibodies were used. In both experiments incubations were at one hour and overnight. The immunostained sections were processed for electron microscopic examination.

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Ultrastructural Immunocytochemistry of Five Rat Pituitary Adenomas

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600003147 ◽

1980 ◽

Vol 38 ◽

pp. 724-725

Author(s):

D. J. McComb ◽

N. Ryan ◽

E. Horvath ◽

K. Kovacs ◽

E. Nagy ◽

...

Keyword(s):

Pituitary Adenomas ◽

Pituitary Tumors ◽

Electron Microscopic ◽

Electron Microscopic Immunocytochemistry ◽

Transmission Electron ◽

Rat Pituitary ◽

Radiation Induced ◽

Group 5 ◽

Group 2 ◽

Microscopic Techniques

Conventional light and electron microscopic techniques failed to clarify the cellular composition and derivation of spontaneous and induced, intrasellar and transplanted pituitary adenomas in rats (1). In the present work, electron microscopic immunocytochemistry was applied to evaluate five adenohypo-physial tumors using a technique described by Moriarty and Garner (2). Spontaneously occurring pituitary adenomas (group 1) were harvested from aging female Long-Evans rats. R-Amsterdam rats were treated with 2 x 1.0 mg estrone acetate (HogivaI) s.c. weekly for 6 months. Pituitary adenomas in excess of 30 mg were removed from these animals to make up the tumors of group 2. Groups 3 and 4 consisted of estrogen-induced autonomous transplan¬ted pituitary tumors MtT.WlO and MtT.F4. Group 5 was a radiation-induced transplanted autonomous pituitary tumor MtT.W5. The tumors of groups 3,4 and 5 were allowed to proliferate in host rats 6-8 weeks prior to removal for processing. Tissue was processed for transmission electron microscopy (glutaraldehyde fixation, OsO4 postfixation and epoxy resin embedding), and electron microscopic immunocytochemistry (3% paraformaldehyde fixation and Araldite embedding).

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