Immuno gold staining (IGS) for electron microscopical demonstration of glial fibrillary acidic (GFA) protein in LR white embedded tissue

Post-embedding immunocytochemical staining methods using gold have so far failed to label intermediate filament antigens in situ in epon or araldite embedded tissue. We have now applied the post-embedding immuno gold staining (IGS) technique for LR White embedded tissue. Glial fibrillary acidic (GFA) protein immunoreactivity was clearly demonstrated electron microscopically on astrocytic filaments of rat cerebellum in situ.     

Immunogold electron microscopic localization of glial fibrillary acidic protein (GFAP) in neurohypophyseal pituicytes and tanycytes of the Mongolian gerbil (Meriones unguiculatus)

The post-embedding immuno gold staining (IGS) technique was used for the ultrastructural localization of glial fibrillary acidic protein (GFAP) in pituicytes and tanycytes of the neurohypophysis. IGS was applied to LR White embedded neurohypophyseal tissue of the Mongolian gerbil (Meriones unguiculatus), a species which contains abundant GFAP-positive pituicytes and tanycytes. GFAP-immunoreactivity could be demonstrated on pituicytic intermediate filaments (IF's) in situ. Thus, it was shown that pituicytes contain GFAP in its filamentous form, what had been a matter of speculation. At the ultrastructural level, gerbil tanycytes and tanycyte-like cells in the external zone of the median eminence were characterized by a great amount of densely packed IF's, which were labeled by both GFAP- or vimentin-antibodies. Sequential immunostaining of serial semithin sections with GFAP- and vimentin-antibodies revealed an invariable coexpression of the two IF proteins in somata and processes of these median eminence cells.     

Immunogold electron microscopic localization of glial fibrillary acidic protein (GFAP) in neurohypophyseal pituicytes and tanycytes of the Mongolian gerbil (Meriones unguiculatus)

Summary The post-embedding immuno gold staining (IGS) technique was used for the ultrastructural localization of glial fibrillary acidic protein (GFAP) in pituicytes and tanycytes of the neurohypophysis. IGS was applied to LR White embedded neurohypophyseal tissue of the Mongolian gerbil (Meriones unguiculatus), a species which contains abundant GFAP-positive pituicytes and tanycytes. GFAP-immunoreactivity could be demonstrated on pituicytic intermediate filaments (IF's) in situ. Thus, it was shown that pituicytes contain GFAP in its filamentous form, what had been a matter of speculation. At the ultrastructural level, gerbil tanycytes and tanycyte-like cells in the external zone of the median eminence were characterized by a great amount of densely packed IF's, which were labeled by both GFAP- or vimentin-antibodies. Sequential immunostaining of serial semithin sections with GFAP- and vimentin-antibodies revealed an invariable coexpression of the two IF proteins in somata and processes of these median eminence cells.     

Cell-free synthesis of glial fibrillary acidic protein

Glial fibrillary acidic (GFA) protein has been synthesized in an RNA-dependent cell-free system derived from rabbit reticulocytes. The cell-free synthesized product appears to have the same size as GFA protein isolated from bovine spinal cord, thus showing that GFA protein does not undergo detectable proteolytic processing.     

Specificity of the glial fibrillary acidic protein for astroglia.

Glial fibrillary acidic protein (GFA) is the main constituent of glial filaments and the close similarity of GFA and neurofilament protein has been recently reported. However, the immunofluorescence staining of peripheral nerve which may be observed with GFA antisera is not due to cross-reaction between GFA and neurofilament protein. Staining of peripheral axons was also observed with control sera obtained by injecting the rabbits with nonimmunogenic GFA preparations isolated with the same procedure. Immune GFA antisera and control sera reacted with sodium dodecyl sulfate extracts of sciatic nerve. However, the precipitin line formed with peripheral nerve crossed the line against GFA protein, thus indicating nonidentity between the two antigens. Buffer extract of sciatic nerves that had been incubated with spinal cord reacted by immunodiffusion with GFA antisera, thus indicating that redistribution of GFA occurred under these conditions.     

Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides

Abstract. A panel of 10 mouse monoclonal antibodies specific for glial fibrillary acidic protein (GFA) has been isolated using porcine GFA as antigen. Although all antibodies recognize GFA purified from porcine spinal cord in the western blot technique, they can be subdivided into at least three groups on the basis of their reactivity against defined fragments of the molecule. Immunofluorescence staining patterns with the monoclonal antibodies performed on tissues and cell lines resemble those reported with conventional polyclonal antibodies directed against GFA. In particular astrocytes and Bergmann glia are strongly stained. In addition mouse monoclonal antibodies specific for either the 200 kd, or the 160 kd, or the 68 kd neurofilament triplet protein have been isolated and characterized. These antibodies are specific for neuronal cells and support conclusions made with similar antigen affinity-purified polyclonal antibodies. The combined set of monoclonal antibodies seems a valuable tool to characterize the different cell types of the nervous system.     

Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides

A panel of 10 mouse monoclonal antibodies specific for glial fibrillary acidic protein (GFA) has been isolated using porcine GFA as antigen. Although all antibodies recognize GFA purified from porcine spinal cord in the western blot technique, they can be subdivided into at least three groups on the basis of their reactivity against defined fragments of the molecule. Immunofluorescence staining patterns with the monoclonal antibodies performed on tissues and cell lines resemble those reported with conventional polyclonal antibodies directed against GFA. In particular astrocytes and Bergmann glia are strongly stained. In addition mouse monoclonal antibodies specific for either the 200 kd, or the 160 kd, or the 68 kd neurofilament triplet protein have been isolated and characterized. These antibodies are specific for neuronal cells and support conclusions made with similar antigen affinity-purified polyclonal antibodies. The combined set of monoclonal antibodies seems a valuable tool to characterize the different cell types of the nervous system.     

Synthesis of Glial Fibrillary Acidic Protein in Rat C6 Glioma in Chemically Defined Medium: Cyclic AMP-Dependent Transcriptional and Translational Regulation

Glial fibrillary acidic protein (GFA) expression was induced in rat C6 glioma in chemically defined medium by the addition of N6, O2'-dibutyryl cyclic AMP (dbcAMP). Induction was dependent on the increase in intracellular cyclic AMP (cAMP), which was linearly correlated with added dbcAMP. Contrary to GFA mRNA synthesis, which can be obtained by cAMP-dependent and -independent pathways, translation of mRNA into GFA was observed only above a cellular cAMP concentration of approximately 0.2 fmol/cell. dbcAMP stimulation did not affect the vimentin concentration, which remained at a low level, but changed the cellular morphology from a bipolar to a stellate shape. A similar morphological change was observed after stimulation of C6 with lipopolysaccharide (LPS). However, LPS did not significantly increase the intracellular concentration of cAMP and the LPS-induced mRNA was not translated into GFA. Our results indicate that GFA synthesis is regulated at the mRNA level and at the translational level and that a cAMP-dependent mechanism determines the ultimate synthesis of GFA by a yet unknown mechanism.     

In vivo and in vitro differentiation of neurons and astrocytes in the rat embryo. Immunofluorescence study with neurofilament and glial filament antisera

Antisera raised against neurofilament (NF) peptides and glial fibrillary acidic protein (GFA) (subunit of glial filaments) have been used to identify neurons and astrocytes in order to study their development and differentiation in rat embryo. In vivo observations showed that NF-positive cells first appeared in 12-day-old embryos, whereas GFA-positive cells appeared in brain and spinal cord on the 18th day. In vitro observations showed that NF-positive cells could be obtained only in cultures from 12-day embryos onwards. The further differentiation of neurons involved neurite elongation, aggregation of cell bodies to form islets, and emergence of very brightly staining prominent neurons with large cell bodies and long neurites which took part in complicate pattern formation. GFA-positive cells appeared in vitro on the 16th day and they could be observed even in cultures obtained from 10-day-old embryos. As the culture aged, the GFA staining became highly fibrillary. There was no physical interaction between neuronal and glial processes.     

Distribution of myosin and the glial fibrillary acidic protein (GFA Protein) in rat spinal cord and in the human frontal cortex as revealed by immunofluorescence microscopy

Summary The glial fibrillary acidic (GFA) protein and myosin were localized in rat spinal cord and human frontal cortex using specific antibodies against GFA protein from human spinal cord and highly purified smooth myosin from chicken gizzard by means of an indirect immunofluorescence microscopical approach. A strong GFA protein and myosin immunoreactivity was found in astrocytes of the white and grey matter and in the external glial limitans membrane. The very fine branches of astrocytic processes stained with antiGFA protein, but not with anti-myosin. Similar results were obtained with the human frontal cortex, where myosin antibodies failed to reveal the very fine branches of protoplasmic astrocytes.As a whole, staining with the GFA protein antiserum was more crisp than with the myosin antibody.Thanks are due to Professor J.R. Wolff, Max-Planck Institute for Biophysical Chemistry, Göttingen, for stimulating discussions, to Ursula König, Christa Mahlmeister and Renate Steffens for skilful technical assistance, and to Heidi Waluk for the photographic workSupported by grants from Deutsche Forschungsgemeinschaft (Br 634/1, Dr 91/1, Un 34/4, Ste 105/19)Dedicated to Prof. Dr. med. H. Leonhardt on the occasion of his 60. birthday     

Ultrastructural and immunohistological characterization of a cell culture model for the study of neuronal-glial interactions.

The differentiation of reaggregating cell cultures of dissociated cerebellar cells from 3- and 6-day-old mice was analyzed by electron microscopy of and immunofluorescence to the glial fibrillary acidic protein (GFA) at intervals between 8 hr and 20 days in vitro. Aggregates in culture for 8 hr were composed of 8–12 undifferentiated cells that were negative for the GFA protein and indistinguishable from each other by electron microscopy. Some cells with extended processes and a few immunofluorescent cells had already appeared after 24 hr in vitro, and the elaboration of a complex neural ultrastructure was observed during the subsequent days. After 10 days in vitro the interior of the aggregate was occupied principally by neurons, the majority of which were granule neurons, and regions of neuropil containing many synaptic complexes. Glial fibers with intense immunofluorescence to GFA were present throughout the aggregates but were mainly concentrated at the periphery. Large unidentified cells protruded from the surface. The subsequent days in culture evidenced a decline in the neuronal character of the aggregates with a concomitant increase in fibrous neuoglia. We suggest that factors controlling neuronal-glial interactions and fibrous gliosis are amenable to analysis in this tissue culture system.     

Immunoperoxidase staining of glial fibrillary acidic (GFA) protein polymerized in vitro: An ultramicroscopic study

The unlabeled antibody peroxidase-antiperoxidase (PAP) method of Sternberger et al. has been employed at the ultramicroscopic level to stain filaments polymerized in vitro from aqueous extracts of multiple sclerosis (MS) plaques. The filaments were heavily decorated with antiserum to the glial fibrillary acidic (GFA) protein but not stained with serum absorbed with GFA protein, preimmunization serum, or anti-rat brain tubulin antiserum. Reassembled rat brain tubulin was heavily stained with antiserum to tubulin but was not stained with antiserum to the GFA protein. The present study provides direct morphological evidence that filaments polymerized in vitro from extracts of MS plaques contain the GFA protein.     

Formation of 100 A filaments from purified glial fibrillary acidic protein in vitro.

Glial fibrillary acidic protein, which is specific to astroglia in the central nervous system, polymerizes in vitro into filaments similar to native ~ 100 Å filaments. Following purification from aqueous extracts of bovine brain by immunoaffinity chromatography, GFA ² protein is highly soluble in very low ionic strength solutions. Sedimentation equilibrium analysis of protein solutions in prefilament solvent conditions (2 mm-Tris · HCl, pH 7.8, 20 °C, containing 0.5 mm-dithiothreitol) indicates a paucidisperse mixture of species in solution with a typical range of apparent weight-average molecular weights from about 186,000 to 227,000. Between pH 6.0 and 8.0 the solubility is a function of pH and ionic strength as well as temperature, and precipitation is favored by lowering the pH or temperature and by raising the ionic strength. GFA protein associates in the form of filaments over a narrow range of pH and ionic strength; optimal conditions for polymerization of a 0.1 mg/ml protein solution are 100 mm-imidazole-HCl buffer (pH 6.8), at a temperature of 37 °C, and there is no requirement for co-factors. Filaments appear primarily as tangles of smooth curvilinear structures approximately 100 Å in diameter and of indefinite length, although some lateral association of filaments into thick bundles is also observed. While the formation of filaments is not affected by the presence or absence of reducing agent, under oxidizing conditions disulfide linkages form between protein subunits. Disassembly is achieved by dialysis against 2 mm-Tris · HCl buffer (pH 8.5), but this process is significantly enhanced by the addition of 0.5 mM-dithiothreitol during assembly and disassembly.These experiments clarify the role of GFA protein as the subunit of astroglialspecific intermediate filaments. In addition, they suggest that the 100 Å filament, as other components of the cytoskeleton, may assemble and disassemble in the glial cytoplasm.     

Immunohistological localization of desmin, the muscle-type 100 A filament protein, in rat astrocytes and Müller glia

The distribution of glial fibrillary acidic (GFA) protein and desmin was compared in cryostat sections of rat brain, spinal cord, and eye by immunofluorescence and peroxidase-antiperoxidase (PAP) staining. Desmin antisera were raised to antigen purified from chicken gizzard. In rat brain and spinal cord, GFA protein and desmin were selectively localized in astrocytes. Neurons and axons were not stained. The only difference between GFA and desmin antisera was the staining of smooth muscle in small arteries with anti-desmin. It was only in retinal glia that a difference in the localization of the two proteins was apparent. As previously reported, only the glia limitans on the inner surface of the retina was demonstrated with GFA antisera in the normal eye. With anti-desmin Müller fibers spanning the whole thickness of the retina were stained. It is concluded that GFA and desmin form two distinct systems of 100 A filaments in astroglia, as previously reported for GFA and vimentin.     

Study of Glial Fibrillary Acidic Protein in a Human Glioma Cell Line Grown in Culture and as a Solid Tumor

Abstract: A continuous human glioma cell line grown in culture and as a solid tumor was analyzed for glial fibrillary acidic (GFA) protein. This material provided a rich source for GFA protein that could also be manipulated and controlled. Immunoperoxidase staining at the light and electron microscopic levels revealed that the cell culture and tumor specimens were strongly positive for GFA protein. When aqueous soluble fractions of the cell culture and tumor were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, electroblotted onto nitrocellulose and stained immunochemically, they contained exclusively low molecular weight (41–43 K-dalton) GFA peptides. SDS (0.15%)-soluble fractions contained either low molecular weight only (culture) or a mixture of peptides ranging from 41 to 49K daltons. SDS (1%) extracts of either cell culture or tumor contained only 49K dalton GFA protein. Two-dimensional gel separation revealed that the GFA protein extracted from either the culture or tumor with 1% SDS resolved to two or three spots at pH 5.8. Low molecular weight GFA peptides (<49K daltons) in aqueous and 0.15% SDS-soluble extracts became increasingly more acidic with decreasing molecular weight. The extremely rapid degradation seen suggests that this cell line may be a valuable system for further study of intermediate filament protein turnover.     

Characterization of rodent pineal astrocytes by immunofluorescence microscopy using a monoclonal antibody (J1-31)

Summary In previous studies pineal astrocytes have been characterized immunohistochemically mainly by use of antisera to glial fibrillary acidic protein. Because of the recent demonstration of this protein in non-astrocytic cells the question of its specificity as an astrocytic marker has been raised. A possible alternative tool for characterizing pineal astrocytes is the J1-31 monoclonal antibody, which is directed against a 30 000 dalton astrocytic protein clearly distinguishable from glial fibrillary acidic protein. Immunofluorescence microscopy of this antibody in the pineal gland of rat and guinea-pig revealed a staining pattern similar to that obtained by glial acidic fibrillary protein antisera. In the rat, J1-31-immunoreactive cells and processes were concentrated in the transitional region between the superficial pineal gland and pineal stalk. Fibrillar J1-31-immunoreactive structures were seen in the most proximal part of the guinea-pig pineal gland. The J1-31 monoclonal antibody therefore appears to be a useful tool for the demonstration of pineal astrocytes; it avoids the specificity problems of glial fibrillary acidic protein immunohistochemistry.Supported by the Deutsche Forschungsgemeinschaft, grant Schr 283/2-1, NSERC (A 5021) and MSI Foundation     

SOD-1 inhibits FAS expression in cortex of APP transgenic mice

Peptides derived from proteolytic processing of the amyloid precursor protein (APP) are important for the pathogenesis of Alzheimers disease (AD). In the present study, we found that transgenic mice overexpressing wild-type human APP gene (hAPP/+) displayed a much higher expression of FAS, one of the death receptor subfamily. This FAS overexpression was significantly reduced in the cortex of mice overexpressing both wild-type hAPP gene and wild-type human superoxide dismutase-1 gene (hSOD-1). Moreover hSOD-1 transgenic expression was associated with an increase of Glial fibrillary acidic protein (GFAP) production. This study indicates that SOD-1 overexpression can inhibit FAS expression, which may be beneficial in AD.     

Immunological and chemical characterization of hamster brain polyribosomes-cytomatrix complexes

We have studied the cytoskeletal nature of a brain subcellular fraction previously shown to contain polyribosomes. We have identified the major proteins of this fraction by electrophoretic comparison to a standard cytoskeletal fraction and by immunodetection. These methods have shown the presence of actin, glial fibrillary acidic protein, and neurofilament triplet proteins. We have also studied the effect of various ions and nonionic detergents on the stability of this structure. It was stable in presence of Triton X-100 up to 2% but disrupted by 200 mM K⁺ acetate.Abbreviations CMT cytomatrix - CSK cytoskeleton - DOC sodium deoxycholate - DTT dithiothreitol - EGTA ethylenglycolbis (-Ether)-N,N-N-N-Tetraacetic Acid - GFAP glial fibrillary acidic protein - PR polyribosome - PRCMC polyribosomes-cytomatrix complex     

Developmental expression of the Glial fibrillary acidic protein (GFAP) gene in the mouse retina

1. In the nervous system, Glial fibrillary acidic protein (GFAP) is a well-known, cell type-specific marker for astrocytes. 2. In the mammalian retina, Muller cells, the major class of retinal glia, do not express GFAP or contain only low amounts of this protein. In retinas with photoreceptor degeneration, however, high levels of GFAP are found. It is possible that GFAP synthesis in these retinas could result from "dedifferentiation" of Muller cells as a consequence of disruption of normal neuron-glia interactions. 3. We have carried out immunocytochemical and in situ hybridization studies to examine whether GFAP or its mRNA is expressed by retinal cells early in embryonic development. 4. Our results show that GFAP-containing cells, which are probably astrocytes, are found only in the ganglion cell and nerve fiber layers and that these cells appear after postnatal day-1 (P-1) and continue to form until P-10. 5. Astrocyte formation starts from the optic disc and moves toward the periphery of the retina at a rate of approximately 160-200 microns per day. 6. An unexpected result from these studies is that GFAP mRNA levels are high in the first week of birth and decline rapidly as the animal develops. 7. Finally, we did not find either GFAP or GFAP mRNA in retinal cells other than astrocytes during normal development.     

Immuno gold staining (IGS) and immuno gold silver staining (IGSS) for the identification of the plant pathogenic bacterium Erwinia amylovora (Burrill) Winslow et al.

Summary For the identification of the plant pathogenic bacterium Erwinia amylovora, the immuno gold staining (IGS) and immuno gold silver staining (IGSS) techniques are tested.The IGS and IGSS methods are at least as sensitive an indirect immunofluorescence and require less primary antiserum. Moreover they have the advantage that the prepatrations can be conserved permanently and unchanged.The preparation of the IGS can be observed with transmitted light or — with considerable better result — using epipolarization microscopy.The IGSS method deserves special attention because of its high contrast in normal brigth field microscopy with transmitted light.