Gold toning

Evidence is presented which establishes a quantitative relationship between the conditions of silver impregnation as they are influenced by pH and time, and the amount of gold toning necessary to secure differentiated axonal staining. No variations in the toning process will secure successful staining if the preceding impregnation and development are at fault.     

Differential Staining of Inner Ear Fluids by Protargol

Present day techniques for processing temporal bones involve celloidin embedding. With a few modifications in Bodian's silver staining procedure the celloidin of the endolymphatic spaces stains darker than that of the perilymphatic spaces providing there is no break in the anatomical barrier between them. Essentially the routine procedure of Bodian is used except that metallic copper is omitted from the staining solution, impregnation time is reduced to 3 hr, reduction time is extended to 10 min and no oxalic acid is used for gold toning.     

Recycling procedure for gold chloride used in neurohistology.

A simple method is described whereby pure chloroauric acid (HAuCl4; "brown gold chloride") is recovered from old solutions used for toning silver-stained sections. The procedure can be carried out in any laboratory and 60 to 80% of the gold initially present in a solution is recovered. The recycled product may be used for toning or for the staining of astrocytes by Cajal's method.     

Gold Toning Improves the Visualization of Nucleolar Organizer Regions in Paraffin Embedded Tissues

A modification of the silver colloid technique for staining nucleolar organizer regions in paraffin embedded tissues is described. This modification involves the application of a gold toning step with subsequent gold reduction, if necessary, following incubation of sections in the standard silver colloid solution. Silver stained nucleolar organizer regions (AgNORs) in toned sections are more sharply delineated when compared to untoned controls. in high grade tumors the addition of the toning step results in significantly higher AgNOR counts due to the ability to discriminate more easily individual AgNORs in argyrophilic aggregates within the nucleus. It is recommended, because of enhanced visualization, that this modification of the silver colloid technique be used in studies involving quantification of AgNORs in tissue sections.     

Gold Toning Improves the Visualization of Nucleolar Organizer Regions in Paraffin Embedded Tissues

A modification of the silver colloid technique for staining nucleolar organizer regions in paraffin embedded tissues is described. This modification involves the application of a gold toning step with subsequent gold reduction, if necessary, following incubation of sections in the standard silver colloid solution. Silver stained nucleolar organizer regions (AgNORs) in toned sections are more sharply delineated when compared to untoned controls. in high grade tumors the addition of the toning step results in significantly higher AgNOR counts due to the ability to discriminate more easily individual AgNORs in argyrophilic aggregates within the nucleus. It is recommended, because of enhanced visualization, that this modification of the silver colloid technique be used in studies involving quantification of AgNORs in tissue sections.     

A silver impregnation method for nervous tissue suitable for routine use with mounted sections.

A simple, reliable silver impregnation method for nervous tissue is described for tissues fixed in various fixatives including formalin, Bouin, and Susa. Sections are impregnated in a solution containing 1 g Protargol, 2 ml of a 1% Cu(NO3)2 solution, 2 ml of a 1% AgNO3 solution, and 2-4 drops 30% H2O2 in 100 ml distilled water. Sections are impregnated 2-5 days at 37 C and thereafter reduced in a hydroquinone-formalin solution. This is followed by gold toning and subsequent reduction, dehydration and mounting. This method has been found to be very reliable and selective.     

Silver Impregnation of the Golgi Apparatus, with Subsequent Nitrocellulose Embedding

The appearance of silver impregnation of the Golgi apparatus can be enhanced by the use of nitrocellulose as an embedding medium. Fixation of 1.5 mm thick pieces of fresh tissue for 8 hr in: glycine, 1.7 gm; 15% formalin, 100 ml; HNO₃, conc., 0.5 ml, at pH 2.6 followed by rinsing in water, 4 hr in 1.5% AgNO₃, another rinse, and 2 hr reduction in 1.5% hydroquinone in 15% formalin. This staining procedure yields consistently good results for rat, rabbit, and human tissues. Low-viscosity nitrocellulose embedding is done by infiltrating at 56 C in 7% nitrocellulose for 0.5 hr, 15% for 4 hr, and 27% for 1 hr. The nitrocellulose is hardened 2 hr in chloroform, after which, sections as thin as 5 μ can be cut on a sliding microtome. Gold toning and counterstaining can be done with the tissue affixed to the slide. The Golgi apparatus is stained dark brown to black, and there is better preservation of cellular detail than in tissues processed in paraffin.     

A Silver Impregnation Method for Nervous Tissue Suitable for Routine use with Mounted Sections

A simple, reliable silver impregnation method for nervous tissue is described for tissues fixed in various fixatives including formalin, Bouin, and Sum. Sections are impregnated in a solution containing 1 g Protargol, 2 ml of a 1% Cu(NO₃)₂ solution, 2 ml of a 1% AgNO₃ solution, and 2-4 drops 30% H₂O₂ in 100 ml distilled water. Sections are impregnated 4-5 days at 37 C and thereafter reduced in a hydroquinone-formalin solution. This is followed by gold toning and subsequent reduction, dehydration and mounting. This method has been found to be very reliable and selective.     

Suppression of Costaining of Nonnervous Tissue in Protargol Technics

If, in the procedure of staining nerve fibers in mounted paraffin sections with Protargol according to Bodian, the reduction after toning with gold chloride is executed in a solution of 3-6 drops of aniline oil in 100 ml of 50% alcohol instead of in the prescribed oxalic acid solution, the selectivity of the staining of peripheral nerves is increased. This is effected by a reduction in the intensity of the staining of nonnervous tissue elements. However, at the same time the staining of nonnervous tissue is richer in details and consequently more satisfactory from a histological point of view than it is according to the original method of Bodian or the modification of this method by Ziesmer (1951).     

Palladium toning of silver-impregnated reticular fibers

Palladium toning is much less expensive than gold toning. Ten minutes in 0.05% potassium hexachloropalladate in 4 N hydrochloric acid tones silver-impregnated reticular fibers as well as 3 min in 0.2% aqueous gold chloride does. Differences in toning of the background depend on the silver stain.     

A Copper Sulfate-Silver Nitrate Method for Nerve Fibers of Planarians

For staining in toto, planarians are fixed in a mixture of 10 ml of commercial formalin, 45 ml of 95% ethanol and 2 ml of glacial acetic acid. After treatment with 70% ethanol 3-10 days, they are washed in distilled water and immersed in 10% CuSO₄. 5H₂O for 3 hr at 50° C, transferred without washing to 1% AgNO₃ for 1.0-1.5 hr at 50° C; and then developed in: 10 ml of 1% pyrogallol, 100 ml of 56% ethanol and 1 ml of 0.2% nitric acid. Gold toning, 5% Na₂S₂O₃ and dehydration follow as usual. For staining sections, material is fixed in the same fixative, embedded in paraffin and sectioned at 10 μ. After bringing sections to water, they are immersed in 20% CuSO₄. 5H₂O for 48 hr at 37° C; then rinsed briefly in distilled water and placed in 7% AgNO₃ for 24 hr at 37° C. They are washed briefly in distilled water and reduced in: hydroquincne, 1 gm; Na₂SO₃, 5 gm and distilled water 100 ml. Gold toning, followed by 5% Na₂S₂O₃ and dehydration completes the process. Any counterstaining may follow.     

Palladium Toning of Silver-Impregnated Reticular Fibers

Palladium toning is much less expensive than gold toning. Ten minutes in 0.05% potassium hexachloropalladate in 4 N hydrochloric add tones silver-impregnated reticular fibers as well as 3 min in 0.2% aqueous gold chloride does. Differences in toning of the background depend on the silver stain.     

A Rapid Silver Impregnation Method for Nervous Tissue: A Modified Protargol-Peroxide Technic

A rapid, reliable silver impregnation method is described for nervous tissue fixed in formol-saline, Bouin or Sum. Sections are impregnated for 10-15 minutes at room temperature or 37 C in a solution containing 0.5 g Protargol-S, 0.005-0.01 g allantoin, 1 ml of 1% Cu[NO₂]₂, 1 ml of 1% AgNO₃. and 1-2 drops of 30% H₂O₂ in 100 ml distilled water. Thereafter the dons arc reduced in a hydroquinone-formalin solution. This is followed by gold toning and subsequent reduction and mounting. Alternatively. following the first reduction, the silver image can be intensified by placing sections in a silver-allantoin bath which is followed by reduction and mounting. This method is very reliable and selective, making it suitable for general routine and research use.     

A RAPID METHOD FOR SELECTIVE SILVER STAINING OF NERVE FIBRES AND NERVE ENDINGS IN MOUNTED PARAFFIN SECTIONS

Abstract The staining described in this paper fundamentally is performed by two manipulations, namely (1) the treatment of deparaffinized sections for about fifteen minutes in a solution containing 15 per cent silver nitrate, 10 per cent potassium nitrate and 0.05 per cent glycine, and (2) the reduction for one minute in a solution of 1 per cent pyrogallol, 55 per cent ethyl alcohol and a trace (0.002 per cent) of nitric acid. After toning, dehydrating and mounting in the usual manner the sections generally are ready for examinations within less than an hour. The method is available for specimens fixed in the ordinary fixatives but those containing oxidizing metal compounds. The procedure is discussed from a theoretical point of view and some results are shown in photomicrographs.     

Demonstration of Alkaline Phosphatase and Periodic Acid-Schiff Positive Material in the Same Section

Alkaline phosphatase activity and periodic acid-Schiff (PAS) positive material were demonstrated in the same section by producing a gold-toned silver deposit at the sites of enzyme activity, followed by the PAS reaction. The glycerophosphate incubating mixture was modified by substituting AgNO₃ (final concentration about 0.17%) for the magnesium salt, and, after a slightly lengthened incubation, the silver was reduced by means of a fine grain photographic developer. Gold toning was followed by the regular PAS staining procedure. Gold deposits in the sites of enzyme activity were fully resistant to the action of periodic acid.     

Stains recently certified

For staining in toto, planarians are fixed in a mixture of 10 ml of commercial formalin, 45 ml of 95% ethanol and 2 ml of glacial acetic acid. After treatment with 70% ethanol 3-10 days, they are washed in distilled water and immersed in 10% CuSO₄. 5H₂O for 3 hr at 50° C, transferred without washing to 1% AgNO₃ for 1.0-1.5 hr at 50° C; and then developed in: 10 ml of 1% pyrogallol, 100 ml of 56% ethanol and 1 ml of 0.2% nitric acid. Gold toning, 5% Na₂S₂O₃ and dehydration follow as usual. For staining sections, material is fixed in the same fixative, embedded in paraffin and sectioned at 10 μ. After bringing sections to water, they are immersed in 20% CuSO₄. 5H₂O for 48 hr at 37° C; then rinsed briefly in distilled water and placed in 7% AgNO₃ for 24 hr at 37° C. They are washed briefly in distilled water and reduced in: hydroquincne, 1 gm; Na₂SO₃, 5 gm and distilled water 100 ml. Gold toning, followed by 5% Na₂S₂O₃ and dehydration completes the process. Any counterstaining may follow.     

Cytochemistry and morphology of synaptic structures in the cerebral cortex of rat.

Studies have been carried out on the synapses in the cerebral cortex of rat by using impregnation with ethanolic solution of phosphotungstic acid, contrast staining with ruthenium red and impregnation with bismuth iodide, with or without subsequent uranyl acetate and lead citrate staining. It has been established that dense projections are adequately visualized with methods demonstrating basic chemical groups (phosphotungstic acid and bismuth iodide), whereas the synaptic vesicles are stained by techniques demonstrating acid chemical groups (ruthenium red and uranyl acetate and lead citrate). On the basis of these observations a hypothesis is forwarded concerning the mechanisms of migration of synaptic vesicles towards the presynaptic membrane. Measurements of the parameters of the dense projections suggest that the configuration of the presynaptic vesicular grid is not uniform along the presynaptic areas.     

Silver Staining of Insect Central Nervous Systems by the Bodian Protargol Method

Improved fixation of ganglia of the central nervous system of Periplaneta americana and Schistocerca gregaria for silver staining by Power's (1943) modification of the Bodian protargol method is given by alcoholic Bouin aged for at least 40 days at 60° C. During impregnation of sections, increased copper and decreased pH give paler staining, more selective for nerve fibres. Prolonging impregnation from 24 to 48 hours weakens the stain and decreases selectivity. The intensity of the stain depends chiefly upon the amount of unreduced (developable) silver combined with the tissues; selectivity is determined mainly by the number and distribution of the reduced silver particles (‘nuclei’). In development, increased sodium sulphite gives more differentiation, increased hydroquinone gives less. Optimum developer composition depends upon impregnation, and thick sections need more differentiation than thinner ones. Within limits, change in one of the factors that control staining can be balanced by changes in others, but by suitable adjustment of the conditions the result can be varied from almost total staining of nerve fibres, for general neuroanatomy, to highly selective staining for tracing individual fibres.     

鸣鸣蝉(Oncotympana maculaticollisMotsch)调音肌的功能特性

本文给出了鸣鸣蝉调音肌(TMc)的结构及其与发声膜(SM)的连接关系,揭示了TMc的调音功能。 TMc的前、后支分别与SM前缘底面的外、内侧连接,有助于牵拉SM,其纵轴与SM的膜面约成120°角。理论上估计,TMc对SM的向下垂直拉力和沿膜面的向前水平拉力可能分别约为总拉力的87％和50％。 TMc具有重要的调音功能。不仅影响每侧SM产生的2个脉冲列(PT)的脉冲幅值,每个PT中第1和2脉冲幅值平均约下降3—10dB;而且影响SM发声过程的均一性。同时,对鸣声谱中第二陪音的峰值频率的幅值有明显的影响,其13600—13900Hz、15015—15100Hz和16756—17090Hz的幅值分别平均下降约5.9、8.4和16.3dB。     

Peculiarities of the staining of neural tissue with procion yellow]

The application of protion yellow allowed to reveal nerve cells of different size, and their dendrites and axons, bundles of nerve fibres for a considerable length. Staining with protional yellow resembles to impregnation of the nervous tissue with silver salts. Combination of protion yellow with dyes of other groups and silver nitrate impregnation improves the stability of staining.