Ultrastructural localization of DNA in tissue culture cell nuclei by means of enzyme-gold and autoimmune sera-protein A-gold techniques

The post-embedding localization of DNA was investigated in cell nuclei by means of DNase I-gold and autoimmune sera-protein A-gold techniques. Using the former technique, gold particles were found mainly over euchromatin, nucleoli exhibit occasionally high labeling. In the latter technique, the use of the serum binding dsDNA confined the label mainly to condensed chromatin.     

Use of enzyme-gold complexes for the ultrastructural localization of hemicelluloses in the plant cell wall

Summary Enzyme-gold complexes have been prepared with an endo 14 xylanase (EC 3.2.1.8) and a 14 mannanase (EC 3.2.1.78). The complexes were applied to ultrathin sections of plant cell walls for the ultrastructural localization of xylans in different tissues of a graminea and for the localization of glucomannans in the tracheids of spruce wood. The method proved to be highly specific and gave a very good contrast of the substrate polysaccharides. Used in conjunction with other cytochemical staining the enzyme-gold labelling provided information about the relative distribution of pectic polymers and xylans in primary walls.     

Use of enzyme-gold complexes for the ultrastructural localization of hemicelluloses in the plant cell wall

Enzyme-gold complexes have been prepared with an endo beta 1----4 xylanase (EC 3.2.1.8) and a beta 1----4 mannanase (EC 3.2.1.78). The complexes were applied to ultrathin sections of plant cell walls for the ultrastructural localization of xylans in different tissues of a graminea and for the localization of glucomannans in the tracheids of spruce wood. The method proved to be highly specific and gave a very good contrast of the substrate polysaccharides. Used in conjunction with other cytochemical staining the enzyme-gold labelling provided information about the relative distribution of pectic polymers and xylans in primary walls.     

Ultrastructural localization of mannoside residues on tissue sections: comparative evaluation of the enzyme-gold and the lectin-gold approaches

Mannoside residues were revealed at the ultrastructural level in different cellular and extracellular compartments by means of the enzyme-gold and the lectin-gold approaches. For the enzyme-gold technique, an alpha-mannosidase-gold complex was prepared and conditions for the preparation of this complex as well as for its application were determined. Labeling was found over the rough endoplasmic reticulum mainly at the level of the membranes, the lumen of the cisternae being devoid of labeling. In the nucleus, the dense chromatin and the edge of the fibrillar threads in the nucleolus were intensely labeled. Few gold particles were present over the Golgi apparatus and mitochondria. The secretory granules in pancreatic cells, the peroxisomes in liver and the mucin in duodenal goblet cells were devoid of labeling. In the extracellular space, the basal lamina was labeled. Over the glomerular basal lamina, the labeling was mainly towards the epithelial side, in close contact with the podocytes. The results with the concanavalin A horseradish peroxidase (Con A-HRP)-gold technique were similar to those found with the enzyme-gold approach. Some differences were, however, detected at the level of the rough endoplasmic reticulum and the nucleus. In the endoplasmic reticulum, Con A-HRP-gold labeling was present over both the membranes and the lumen of the cisternae. In the nucleus, the labeling was mainly over the dispersed chromatin. These differences may be due to the binding of Con A not only to mannoside but also to other sugar residues as well as to the affinity of HRP-gold for some nucleoplasmic components.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural localization of beta-D-galactan in the nuclei of the myxomycete Physarum polycephalum.

The acellular slime mold Physarum polycephalum produces an extracellular sulfated and phosphorylated beta-D-galactan which was recently isolated from the nuclei of this organism. This polysaccharide has now been localized in the nuclei of P. polycephalum by electron microscopy using a specific "sandwich" technique: thin sections of P. polycephalum microplasmodia were incubated with the Ricinus communis lectin specific for D-galactose residues. The bound lectin was then localized with gold granules labeled with a galactose-terminated glycoprotein (desialylated ceruloplasmin). The galactan was found in the nuclei mainly associated with chromatin and, also, but to a smaller extent, in the cytoplasm and in some vacuoles. The specificity of the method was assessed by marking under the same condition the galactomannan present in the cell wall of the yeast Schizosaccharomyces pombe.     

Ultrastructural localization of intracellular antigens by the use of protein A-gold complex.

An immunocytochemical technique for the demonstration of intracellular antigens (secretory proteins) on thin sections is reported. Staphylococcal protein A which reacts with the Fc fragment of IgG molecules was labeled with colloidal gold as a marker. The antigenic sites were visualized on aldehyde-fixed and Epon-embedded tissue in a two step procedure. The specific antisera were applied to thin sections for binding to the antigens and then visualized by the protein A-gold complex. By using this technique different secretory proteins of the exocrine and endocrine pancreas were localized. The protein A-gold technique is proposed as a general method for visualization of antigenic sites on thin sections.     

Electron microscopical localization of nucleic acids by means of nuclease-gold complexes

Summary Nucleic acids can be specifically localized at the electron microscope level by means of enzyme-gold complexes. Two enzymes RNAase A and DNAase I were labelled with colloidal gold, and the enzyme-gold complexes obtained applied on thin sections of glutaraldehyde-fixed and Epon-embedded tissues. Using RNAase-gold, the rough endoplasmic reticulum and the nucleolus of different cells appeared densely labelled. With the DNAase-gold the labelling was present over the euchromatin and the mitochondria. The quantitative evaluation, performed on different cellular compartments of the pancreatic acinar cells, confirmed the qualitative observations and ascertained the specificity of the labelling. The application of this technique, for the demonstration of nucleic acids in different tissues, is illustrated.     

Ultrastructural immunocytochemical localization of excitatory amino acids in the somatosensory system.

We studied the ultrastructure and the synaptic arrangement of glutamate-immunoreactive terminals in rats, in the superficial laminae of the spinal cord, the brainstem cuneate nucleus, and the thalamic ventroposterolateral nucleus, where a role for glutamate as neurotransmitter has been suggested by biochemical, physiological and pharmacological approaches. The antiserum employed was raised against glutaramate conjugated to keyhole limpet hemocyanin with glutaraldehyde, and was used for pre-embedding staining with an avidin-biotin-peroxidase method and for post-embedding staining with an immunogold procedure. Both methods yielded similar results, consisting of labeling of selected terminals in all the areas examined. Double immunogold labeling on the same thin section using antisera against gamma-amino-butyric acid (GABA) or substance P (SP), in combination with the anti-glutamate serum, showed that staining for glutamate and GABA was present in different terminals in all the regions examined; glutamate and SP were co-localized in a few terminals only in the superficial laminae of the spinal cord. By performing immunogold staining in combination with anterograde tracing, glutamate immunoreactivity could be localized in identified primary afferents to the dorsal spinal cord and cuneate nucleus, and in lemniscal afferents to the thalamus.     

Ultrastructural localization of β-galactan in the nuclei of the myxomycete Physarum polycephalum

The acellular slime mold Physarum polycephalum produces an extracellular sulfated and phosphorylated β-D-galactan which was recently isolated from the nuclei of this organism. This polysaccharide has now been localized in the nuclei ofP. polycephalum by electron microscopy using a specific “sandwich” technique: thin sections of P. polycephalum microplasmodia were incubated with the Ricinus communis lectin specific for D-galactose residues. The bound lectin was then localized with gold granules labeled with a galactose-terminated glycoprotein (desialylated ceruloplasmin). The galactin was found in the nuclei mainly associated with chromatin and, also, but to a smaller extent, in the cytoplasma and in some vacuoles. The specificity of the method was assessed by marking under the same condition the galactomannan present in the cell wall of the yeast Schizosaccharomyces pombe.     

Ultrastructural localization of cell wall teichoic acids in Streptococcus faecalis by means of concanavalin A

Some teichoic acids are known to be partially substituted by α-D-glucopyranosyl residues such as the teichoic acids of Streptococcus faecalis NCIB 8191. They will, therefore, bind specifically the phytohemagglutinin concanavalin A. Concanavalin A labelled with mercury or colloidal gold coated with concanavalin A has been used to mark isolated cell walls in order to localize the teichoic acids at the ultrastructural level. Besides these two direct marking techniques, the indirect concanavalin A-peroxidase technique (localization of peroxidase by the diaminobenzidine method followed by postosmication) has been applied to thin sections of premarked cells. All three methods gave almost identical results, namely, a dense and homogeneous distribution of the cell wall teichoic acids. In control experiments total inhibition was achieved in the presence of methyl-α-D-mannopyranoside. After trichloroacetic acid or alkali extraction of the teichoic acids from isolated walls no marking could be detected.     

Ultrastructural localization of DNA and RNA in Allium porrum interphase cells by means of nuclease-gold complexes

Summary Nucleic acids have been localized inAllium porrum interphase meristematic cells by means of labelling with nuclease-gold complexes, a technique which provides high resolution and improved specificity. DNase-gold labelling was observed over dense chromatin and to a lesser extent over dispersed chromatin. Nucleolar labelling was restricted to the dense fibrillar component, very few particles being located over the fibrillar centres. Labelling by the RNase-gold complex was present over both the cytoplasm and the nucleoplasm. Cytoplasm labelling was intense over the rough endoplasmic reticulum but absent over vacuoles. In the nucleoplasm many gold particles were located at the border between the condensed and the dispersed chromatin. Nucleolar labelling was intense over the granular zones but many gold particles were also seen over the dense fibrillar component. Fibrillar centres showed, however, no labelling with the RNase-gold complex. These results are consistent with previous autoradiographic and cytochemical observations carried out on the same plant material.     

Ultrastructural localization of cholesterol by enzyme histochemistry

Summary The histochemical localization of cholesterol using oxidized diaminobenzidine as the final reaction product was studied at the electron microscopical level and compared with the digitonin method of cholesterol localization based on cholesterol digitonide as the final reaction product. Tissue chopper sections of fixed rat adrenal glands were incubated at 37° C in a medium consisting of 0.8 units/ml cholesterol oxidase, 1.4 units/ml cholesterol ester hydrolase, 50 units/ml horseradish peroxidase, 0.5 mg/ml diaminobenzidine, 0.1% v/v Triton X-100 (or Surfal) and an endogenous peroxidase inhibitor in 0.1m phosphate buffer, pH 7.0. An electron-dense osmiophilic reaction product was observed in many lipid droplets, intracellular vesicles and focally around mitochondria. Appropriate control experiments indicated that deposition of reaction product depended on the presence of cholesterol and the necessary enzymes. Comparison studies using digitonin confirmed the presence of cholesterol in the lipid droplets, but ultrastructural distortion limited the resolution of the more discrete deposits of cholesterol such as around mitochondria. The enzyme method permits finer resolution of these discrete deposits of cholesterol than the digitonin method because it does not cause distortion of cellular ultrastructure attributed to the formation of cholesterol digitonide. The enzyme method or a combination of enzyme and digitonin enables localization of free, esterified or total cholesterol.     

Ultrastructural localization of serotonin in the intrapulmonary neuroepithelial bodies of neonatal rabbits by use of immunoelectron microscopy

Summary The ultrastructural localization of serotonin in neuroepithelial bodies (NEB) of neonatal rabbits was examined by use of the recently developed immunogold-staining method. In the granulated epithelial cells of NEB serotoninimmunoreactive material could be demonstrated only in dense-cored vesicles (DCV) of the first type. No immunoreactivity was observed in DCV of the second type. The present findings support the previously made suggestion that the corpuscular NEB cells contain two distinctive DCV types which react differently with the immunogold-staining procedure for identifying serotonin. NEB, known as intrapulmonary sources of serotonin (and peptide substances), may play a prominent role in lung physiology.