A silver impregnation technique for the study of steroid receptors in central nervous system tissue prepared for autoradiography

The commonly used silver stains were found to be unsatisfactory for nervous tissue processed for autoradiography. A silver impregnation procedure for central nervous system tissues prepared for the autoradiographic study of steroid receptors is described. The procedure is a combination of several silver and reticular strains made up in solutions containing dimethylsulfoxide. The technique clearly distinguishes perikarya of neurons, brain nuclei and fiber tracts without substantial loss of silver grains, and thus greatly facilitates the identification of steroid receptor nuclei at all levels of the central nervous system.     

A silver impregnation technique for normal axons in the human central nervous system for celloidin and epon sections, with substitutes for soft tap water

An improved silver technique has been developed for human CNS axons in sections from celloidin blocks that resist impregnation because of prolonged storage in alcohol. This method also gives consistently good impregnation of recently fixed material, and thus is suitable for routine use. Slightly modified, the method is also successful with osmicated Epon embedded sections. The quality of silver impregnation in methods using tap water in the reducing solutions varies in different laboratories. Having established that hard water is essential, substitutes for soft water were sought and found.     

A silver impregnation method for embedded central nervous tissue of the rat

A simple and yet reliable silver impregnation method, using potassium ferrocyanide, for demonstrating nervous tissue of the rat central nervous system embedded in paraffin or paraplast is described. The method reported here is compared and discussed with earlier techniques using potassium dicyanoargentate, potassium ferrocyanide and potassium ferricyanide.     

Rapid method of silver nitrate impregnation of elements of the peripheral nervous system suitable for celloidin and paraffin sections

According to the method of neural elements impregnation in the authors' modification, the object is fixed for 6-12 h in Lillie fluid cooled to 4 degrees C. Then the object is kept under tap water for 2-6 h. Frozen sections are prepared and kept in pure pyridine for 1-6 h. When the sections are embedded into paraffin or celloidin, they are put into alcohol solutions gradually decreasing their concentration until water is reached, then put into pyridine. In order to remove cellulose, the celloidin sections are treated in 3 portions of pyridine (in the 1st and 2nd-for 10 min, and in the 3d-for 6 h). Then they are washed under tap water for 2-4 h and in distilled water for 30-40 min. Further treatment is performed according to the methods by Bielschowsky - Gros, Kampos or Rasskazova. Excess silver is removed by treating the sections in 2% ammonium persulfate under the microscope control (the process is stopped by putting the sections into 7% sodium hyposulfate for 10 min). Then the sections are treated in 0.1% aurum chloride, in 5% hyposulfite to reveale the tissue background [corrected] and by means of routine histological techniques either after Brashet, Hale, PAS-positive reaction or other methods applied after fixation in Lillie fluid.     

A new fixative solution to precede the reduced silver impregnation of arthropod central nervous systems.

Arthropod central nervous tissue is fixed for 1 hr at 20 C in 8% pure formic acid in 1:1 n-butanol/n-propanol prepared immediately before use (FBP), then washed for 15-30 min in 90% ethanol, and embedded in paraffin wax. Impregnation is by modified Ungewitter techniques in which the silver bath is preceded by mercury/cobalt mordanting, or by modified Holmes' methods following similar mordanting procedures. The methods yield high resolution of axons with minimal background staining, while the staining of neuronal somata is suppressed. They succeed with brains of crustacea and Odonata and other difficult materials. Tissues fixed in FBP are hard and require care in sectioning.     

Stabilization of silver chromate Golgi impregnation in rat central nervous system neurons using photographic developers. II. Electron microscopy

The silver chromate precipitate present in neurons impregnated according to the Golgi-rapid and Golgi-Kopsch procedures can be stabilized by treatment with a photographic developer. In a complementary light microscopic study the stabilizing properties of various photographic developers were tested. Kodalith, Elon-ascorbic acid, HC-110, D-19 and Neutol proved to be the most successful. In the present electron microscopic study, we studied the distribution, shape and size of the particles found in Golgi-rapid and Golgi-Kopsch-impregnated neurons by treatment with each of these developers and, simultaneously, the effect of the developer on the preservation of the ultrastructural details. The reaction product after developer-treatment of Golgi-rapid material is sufficiently stable to withstand embedding and thin sectioning, whereas in Golgi-Kopsch material additional gold chloride "toning" is necessary. In Golgi-impregnated, Kodalith-, Elon-ascorbic acid-, or HC-110-treated material the formed particles are small and located in the cytoplasm, limited by the plasma membranes of the impregnated profiles. In Golgi-impregnated, D-19 treated neurons, the formed particles are relatively coarse. The majority of these particles are within cytoplasm, but particles may also lie either across or entirely outside the plasma membranes of the impregnated profiles. A large number of the small particles in Golgi impregnated, Neutol-stabilized neurons can be seen partly or entirely outside the plasma membranes of the impregnated profiles. Good original ultrastructural preservation seems to be unaffected by developer treatment. Treatment of Golgi material with sodium bromide before stabilization (bromide substitution) results in the formation of small silver particles both inside and outside the impregnated profiles. The sodium bromide step of this procedure has an adverse effect on the preservation of ultrastructural detail.     

An impregnation technique for studying distribution of mitochondria in central nervous system

Summary An impregnation technique for demonstration of mitochondria in central nervous system has been described. Formol perfused material is being chromated, embedded in paraffin and impregnated with a modified Nauta's method. The reaction is specific since reduced silver particles are deposed within the matrix of mitochondria. Phenomenon of complete and incomplete impregnation is discussed. Mitochondria in perikarya of neurons, dendrites and glia tend to be unstained if the impregnation is incomplete, while axonic and praesynaptic ones are labelled. In neuropil of different regions of the brain various types of mitochondrial aggregates appear in complete impregnation. The method may be a valuable tool for studying brain architecture and synaptology.With a grant of Alexander von Humboldt-Stiftung at the II. Institute of Anatomy, Berlin.     

Stabilization of silver chromate Golgi impregnation in rat central nervous system neurons using photographic developers. I. Light microscopy

Deterioration of Golgi impregnation begins immediately after impregnated tissue blocks are sectioned with the Vibratome. The first signs of deterioration are fading of delicate impregnated processes, the disruption and fragmentation of dendrites, and, eventually, fading of entire neurons. These changes can be prevented by stabilization, i.e., by converting the water soluble silver chromate Golgi precipitate into metallic silver or by replacing the silver with some other dense, insoluble material. A technique is described using photographic developers to treat Vibratome sections containing Golgi-rapid or Golgi-Kopsch impregnated CNS neurons. In this way part of the silver chromate Golgi precipitate is reduced to metallic silver, and the remaining silver chromate is then removed with sodium thiosulfate. Of the various developers tested, Kodalith and Elon-ascorbic acid gave the best results, with excellent stabilization of the most delicate structures, such as the stalks of dendritic spines and finely woven axonal plexuses. Treatment with other developers (HC-110, Neutol, D-19, D-76, D-163, Kodak Universal, Rodinal, Atomal, Diafine, Eukobrom, Microdol-X) resulted in stabilization ranging from good to poor. Good stabilization of Golgi impregnation could also be achieved by first exposing the sections to sodium bromide (bromide substitution) followed by treatment with D-19, Kodalith, Elon-ascorbic acid or HC-110. After stabilization, the sections can be counterstained with aqueous cresyl violet or with alcoholic thionin without degradation of the stabilized Golgi image. The counterstain permits exact determination of the position of impregnated neurons in cortical layers or subcortical nuclei.     

Influence of carbon dioxide on the impregnation of the nervous tissue by silver

It has been shown that 4% carbon dioxide (CO₂) in the air above reaction mixture inhibits the initiation of the formation of silver nanoparticles from complexes with biogenic amines (noradrenaline and serotonin). At the same concentration of CO₂ in the air above solution of AgNO₃, which is used for staining nerve tissues by the method of Golgi, neurons are preferentially stained, whereas at a concentration of 0.06%, vessels are stained. It is suggested that the entry of free silver ions to neurons is due to the inhibition of sites of initiation of silver nanoparticles in vessels at high CO₂ concentrations, while the lack of inhibition leads to silver precipitation in vessels at low CO₂ concentrations. It can be assumed that, for stable silver impregnation, the concentration of CO₂ must be controlled.     

Antibiotic treatment of abscesses of the central nervous system.

Samples of intracranial pus and serum from 32 patients were assayed to determine the concentrations reached in them of penicillin, ampicillin, cloxacillin, cephaloridine, gentamicin, chloramphenicol, fusidic acid, and lincomycin. Metronidazole had not been given. Penicillin penetrated abscesses reasonably well, but other beta-lactam antibiotics did not. The penetration of chloramphenicol was erratic. Aminoglycosides penetrated poorly, but lincomycin and fusidic acid penetrated well. Assay of sulphonamides and co-trimoxazole in pus was unreliable. These studies indicate that treatment of abscesses of the central nervous system should be considered according to the site and the likely antecedent cause. Abscesses of sinusitic origin, usually in the frontal lobe, yield penicillin-sensitive streptococci. Penicillin is the drug of choice. Abscesses of otitic origin, usually in the temporal lobe, yield a mixed flora, often including anaerobic bacteria. Multiple antibiotic therapy is indicated. Abscesses of metastatic or cryptogenic origin yield streptococci or mixed cultures, and multiple therapy is appropriate while awaiting the bacteriological results. Spinal and post-traumatic abscesses yield Staphylococcus aureus, and fusidic acid is the drug of choice.