A silver-reducing component in rat striated muscle. II. Isolated sarcoplasmic reticulum vesicles

In previous work on rat striated muscle cells a silver-reducing component was found selectively localized at the terminal cistern/transverse tubule system (Tandler and Pellegrino de Iraldi 1989). To further investigate that problem we performed the Hg-Ag argentaffin reaction on a sarcoplasmic reticulum fraction from rat skeletal muscle. Circular profiles corresponding to vesicular structures were found outlined by silver grains. The number of silver "stained" vesicles were less than the total number vesicles stained by conventional procedures. The correlation between argentaffinities in the intact muscle fiber and their subcellular organelles indicated that the Hg-Ag reactive vesicles must be those derived from the terminal cisternae of the sarcoplasmic reticulum. The silver-reducing constituent aggregates in the presence of 1 mM CaCl2 or 0.5 M K cacodylate. The state of aggregation induced by Ca2+ was not affected by incubation with 0.5% Triton X-100 or by 2 mM EDTA, thus suggesting a localization at or near the membrane of the terminal cistern vesicle facing the junctional gap. In Laemmli SDS-acrylamide gels the Hg-Ag reaction stained all proteins in a manner similar to Coomasie blue. It is suggested that the selective histochemical staining is the result of differential reactivities due to steric requirements of the chemical reaction.     

Influence of the protein staining in the fast ultrasonic sample treatment for protein identification through peptide mass fingerprint and matrix-assisted laser desorption ionization time of flight mass spectrometry

The influence of the protein staining used to visualize protein bands, after in-gel protein separation, for the correct identification of proteins by peptide mass fingerprint (PMF) after application of the ultrasonic in-gel protein protocol was studied. Coomassie brilliant blue and silver nitrate, both visible stains, and the fluorescent dyes Sypro Red and Sypro Orange were evaluated. Results obtained after comparison with the overnight in-gel protocol showed that good results, in terms of protein sequence coverage and number of peptides matched, can be obtained with anyone of the four stains studied. Two minutes of enzymatic digestion time was enough for proteins stained with coomassie blue, while 4 min was necessary when silver or Sypro stainings were employed in order to reach equivalent results to those obtained for the overnigh in-gel protein protocol. For the silver nitrate stain, the concentration of silver present in the staining solution must be 0.09% (w/v) to minimize background in the MALDI mass spectra.     

Critical Staining of Pancreatic Alpha Granules with Phosphotungstic Acid Hematoxylin

Mammalian pancreatic alpha granules were differentially stained with phosphotungstic acid haematoxylin. Paraffin sections were dewaxed and hydrated, oxidised 5-40 sec in freshly prepared 0.3% KMnO₄ acidified with 0.3% (w/v) H₂SO₄, decolourised in 4% potassium metabisulphite, mordanted 20 min to 2 hr in 4% iron alum, stained in phosphotungstic acid haematoxylin 16-48 hr, rinsed in 95% ethanol until no stain runs from the tissue, dehydrated in absolute ethanol, cleared in xylene, and covered in synthetic resin. Advantages of this procedure are: (1) consistent, reproducible staining; (2) applicability to all the common laboratory mammals and man; (3) wide latitude at each stage, permitting its use as a routine method; and (4) superior visualization of alpha granules, due to suppression of background staining and absence of glare. For fixation, formalin-acetic or Bouin's solution is recommended.     

Selective Silver Impregnation of Degenerating Axons In The Central Nervous System

Rat and rabbit brains containing surgical lesions of 5-10 days' duration were fixed in 10% formalin (neutralized with calcium carbonate) for 1 week to 6 months. Frozen sections (15-20 n) were rinsed and then soaked 7 minutes in a 1.7% solution of strong ammonia in distilled water. Subsequent treatment was as follows: rinse; 0.05% aqueous potassium permanganate 5-15 minutes; 0.5% aqueous potassium metabisulfite, 2 changes of 2.5 minutes each; wash thoroughly in 3 changes distilled water; 1.5% aqueous silver nitrate, 0.5-1.0 hr.; 1% citric acid, 5-10 sec.; 2 changes distilled water; 1% sodium thiosulfate, 30 see.; 3 changes distilled water. Each section is then processed separately. Ammoniacal silver solution (450 mg. silver nitrate in 10 ml. distilled water; add 5 ml. ethanol; let cool to room temperature; add 1 ml. strong ammonia water and 0.9 ml. of 2.5% aqueous sodium hydroxide), 0.5-1.0 min. with gentle agitation. Reduction of about 1 minute is accomplished in: distilled water, 45 ml.; ethanol, 5 ml.; 10% formalin, 1.5 ml.; 1% citric acid, 1.5 ml. Rinsing; 1% sodium thiosulfate, 10 sec.; thorough washing followed by dehydration through graded alcohol and 3 changes of xylene or toluene complete the staining process. Normal nerve fibers are slightly stained to unstained, degenerating fibers, black. The treatment in potassium permanganate is critical since too little favors overstaining of normal fibers and too much abolishes staining of degenerating fibers.     

The Demonstration of Beta Cells in Pancreatic Islets and Their Tumors

The stain is applied routinely to tissues fixed in 10% buffered formalin (pH near 7.0) or in Bouin's fluid. Bring paraffin section to water as usual and mordant 72 hr in 5% CrCl₃ dissolved in 5% acetic acid. Wash in water and in 70% alcohol and stain 6 hr. Formula of staining solution: new fuchsin, 1% in 70% alcohol, 100 ml; HCl, conc., 2 ml and paraldehyde, 2 ml, mixed together and added to the dye solution; let stand 24 hr before use. After staining, wash in running tap water 5-10 min, rinse in distilled water and counterstain if desired. Dehydration in alcohol, clearing and covering completes the process. When the paraldehyde is obtained from a freshly opened bottle, standardized staining times can be used and thus eliminate the necessity of differentiating individual slides. The granules of beta cells stained deep blue to purple and were demonstrated in the pancreatic islet of man, dog, mouse, frog, guinea pig and rabbit.     

A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of proteins after two-dimensional electrophoresis

We report on a new silver stain especially developed for staining large gels (25 cm x 20 cm) from the Hoefer ISO-DALT system for matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of proteins. The staining protocol can be summarized as follows: the gels are sensitised in tetrathionate/potassium acetate solution and washed several times in distilled water. After impregnation with silver nitrate, the silver is reduced in the presence of potassium carbonate, thiosulphate and formaldehyde. The staining procedure is stopped with Tris/acetate after which the gels are rinsed and stored in water before spot picking for MALDI-TOF analysis is performed. This protocol has several advantages over existing ones. The gels are stained in a new apparatus that reduces gel handling to a minimum thus also reducing the contamination with keratins to a minimum. The development times in potassium carbonate are very long (up to 40 min) thus improving batch-to-batch reproducibility. Only the surface of the proteins is stained and the silver can be oxidized, thereafter MALDI-TOF can be performed with protein loads as little as 100 micrograms per gel.     

A simple and economical modification of the Masson-Fontana method for staining melanin granules and enterochromaffin cells

Enterochromaffin cells from the small intestine of man, guinea pig, dog, chicken, rabbit, cat and rat were stained using the Masson-Fontana ammoniacal silver method with varying dilutions of silver nitrate solution (0.25 to 5 g per 100 ml of distilled water) and incubation temperatures (60 C and 75 C). The 0.5% solution of silver nitrate gave an argentaffin pattern similar to that of the 5% solution and had two major advantages: economically, since much less silver nitrate is used, and methodologically, since low background resulted with tissue of those species (rat, cat and rabbit) that required unusually long incubation. The staining of melanocytes was similar for all dilutions at the usual staining time (15-30 min).     

A Simple and Economical Modification of the Masson-Fontana Method for Staining Melanin Granules and Enterochromaffin Cells

Enterochromaffin cells from the small intestine of man, guinea pig, dog, chicken, rabbit, cat and rat were stained using the Masson-Fontana ammoniacal silver method with varying dilutions of silver nitrate solution (0.25 to 5 g per 100 ml of distilled water) and incubation temperatures (60 C and 75 C). The 0.5% solution of silver nitrate gave an argentaffin pattern similar to that of the 5% solution and had two major advantages: economically, since much less silver nitrate is used, and methodologically, since low background resulted with tissue of those species (rat, cat and rabbit) that required unusually long incubation. The staining of melanocytes was similar for all dilutions at the usual staining time (15-30 min).     

Controlled Differentiation of Cells and Connective Tissue Fibers in Silver Staining

Controlled silver staining of connective tissue fibers and sometimes of these fibers and cells simultaneously can be obtained. 1. Fix in 10% formalin. Embed in paraffin and cut sections as usual, but do not mount them on slides. Deparaffinize and hydrate through xylene, alcohols and distilled water and henceforth treat them the same as frozen sections. Real frozen sections can also be used. 2. Treat with a freshly prepared 1% solution of KMnO₄, usually 15-60 sec, sometimes up to 10 min. 3. Wash in distilled water, 5-10 sec. 4. Decolorize in 2% potassium metabisulfite, 10-20 sec. 5. Place in distilled water, 1 min. 6. Sensitize with 2% iron alum, 1 min. 7. Place in distilled water, 1 min. 8. Impregnate in Gomori's silver oxide solution, 2 min. 9. Wash in a 1.5% aqueous solution of pyridine, about 15 sec. 10. Reduce in a mixture containing 0.25% gelatin and 2% formalin 1 min. 11. Repeat steps 7 to 10 once or several times until the connective tissue fibers are completely stained. For cell staining (which may fail) proceed as follows: After the first insufficient staining of the connective tissue fibers, rinse in distilled water, dip for 1 sec in Gomori's solution and reduce immediately in gelatin-formalin without previous washing in pyridined water. This step can be repeated. 12. If the staining is too strong, decolorize as needed in 2% iron alum. 13. Toning in 0.2% gold chloride, 5 min or more, followed by fixation in 5% sodium thiosulfate, 1 min, is optional. Counterstain as desired. 14. Wash in tap water, dehydrate, clear in xylene and mount in balsam. The same technique applied to sections attached to slides gives good results but inferior to that obtained in paraffin sections processed in the loose, unmounted condition.     

Staining Reticulin with Gold

This bromine-iodine-gold chloride-reduction sequence stains reticulin in formalin-fixed paraffin sections without risk of sections becoming detached. After hydration, sections are exposed to 0.2% bromine water containing 0.01% KBr for 1 hr, then rinsed and placed for 5 min in a solution consisting of KI, 2 gm; iodine crystals, 1 gm; and distilled water, 100 ml. After this the sections are well washed in distilled water, immersed for 5 min in 1% w/v aqueous solution of chloro-auric acid, again rinsed in distilled water, and the gold is reduced by placing in freshly made 3% H₂O₂ for 2-4 hr at 37 C, or in 2% oxalic acid for 1-3 hr at the same temperature.     

Silver Staining, Featuring Rapid Reduction, For Whole Mounts of Retina and Optic Pathways in Chick Embryos

Developing and established nerve fibers in the retina and in superficial tracts of the brain can be stained and viewed en bloc. The method was developed on chick embryos of 2 days of incubation to several months post-hatching but could be used on other material provided that the objects of interest were within 35 μ of the surface. Procedure: (1) Place the entire eye or head in 50% pyridine for at least 16 hr. (2) Wash well for 5-7 hr with hourly changes of distilled water or with running tap water for 4-6 hr followed by several changes of distilled water. (3) Transfer to 95% ethanol for 16-48 hr. (4) Impregnate with 1.5% AgNO₃ for 2 days at 37 C. (5) Submerge in water and, when staining the retina, remove the vitreous body and apply an aqueous solution of 0.25% pyrogallic acid in 1.25% formalin by directing a narrow stream of this reducer against the retina for 2-5 sec. Wash the eye with distilled water 30-60 sec after applying the reducer. When staining the brain, remove the meninges under water, direct the stream of reducer against the brain for 20-30 sec, and rinse the brain immediately after the nerves have stained. (6) Dissect the specimen and make temporary mounts in glycerol; or, dehydrate and clear for resin mounting. The technique stains both mature and growing axons with their growth cones and sometimes their cell bodies. The fiber patterns show best on the surfaces of the retina and brain. The stain works consistently and is suited to the study of both normal and abnormal development.     

A Reliable Method for Demonstrating Axonal Degeneration Shortly After Axotomy

A method has been elaborated by which degenerating axons can be selectively impregnated with silver. Based on a reconsideration of the physicochemical mechanisms of the degeneration methods it takes advantage of physical developers over the chemical ones. The staining procedure is applied to frozen sections of brains fixed with formol. It consists of 6 steps: (1) pretreatment with alkaline hydroxylamine, (2) washing in acetic acid, (3) impregnation in silver nitrate in the presence of ferric ions, (4) washing in citric acid, (5) physical development, and (6) washing in acetic acid. By electron microscopy silver precipitates by this method are almost entirely restricted to the cytoplasm of dense, degenerating axons, sparing mitochondria and myelin sheaths. No special expertise is required to achieve reproducible results. Large numbers of sections treated simultaneously, and large sections, can be stained uniformly. Light microscopic criteria are described which help diagnose the source of possible failures. Low background staining allows dark field illumination and television image analysis to be applied. The method works at survival times of only 3 to 5 days after axotomy. Hence, degenerating axons and axon terminals can be stained in alternating sections from the same brain using this method and another being described separately, which, using different conditions, demonstrates degenerating axon terminals.     

Adaptation of the Nauta Method for Use on Degenerating Fibers in Rat Cortex and Thalamus

Lesions produced in the cerebral cortex of rats were studied by Nauta's method for degeneration. The brains were perfused with physiological NaCl solution, followed by 10% neutral (CaCO₃) formalin. The brains were removed and stored in the formalin for 2 wk to 1 yr. Experimental modifications of the staining method showed that its sensitivity for fine degenerating fibers could he enhanced by the following changes: (a) omitting 0.05% potassium permanganate; (b) replacing the hydroquinone-oxalic acid mixture with 0.1% pyrogallol. Procedure: (1) frozen sections to water; (2) 0.5% phosphomolybdic acid, 45 min; (3) distilled water, 1 min; (4) 0.1% pyrogallol (aq.), 2 min; (5) distilled water, 3 washes of 1 min each; (6) 1.5% silver nitrate (aq.), 30 min; (7) distilled water, 1 min, (8) Laidlaw's ammoniated silver carbonate, 10110 sec; (9) Nauta's reducer, 1-2 min; (10) distilled water, 1 min; (11) 1.0% Na₂S₂O₃, 2 min; (12) distilled water 3 changes, 1 min each; (13) dehydrate, clear, and cover. This method gave equally good results on degenerating axons in both cortex and thalamus.     

Rapid Bielschowsky silver impregnation method using microwave heating

The Bielschowsky silver impregnation method has been used extensively to demonstrate neuronal processes including dendrites, axons and neurofibrils. In this study, we examined the differences in the time required for and the staining quality of the Bielschowsky method for neuronal processes when microwave heating was used instead of processing at room temperature. For this purpose, a control group of sections stained according to the conventional method at room temperature was compared to an experimental group stained in a microwave oven at 180 W for 2, 4 and 1 min in 2% silver nitrate, ammoniacal silver nitrate and gold chloride, respectively. Light microscopic examination demonstrated that the normal structure was preserved in both groups and that there was no difference in the staining quality between the control and the microwave groups. In addition, staining time for this procedure was reduced to 8 min by using the microwave oven. Our study revealed that microwave irradiation can be used safely for Bielschowsky silver impregnation of neuronal tissues.     

Rapid Bielschowsky silver impregnation method using microwave heating

The Bielschowsky silver impregnation method has been used extensively to demonstrate neuronal processes including dendrites, axons and neurofibrils. In this study, we examined the differences in the time required for and the staining quality of the Bielschowsky method for neuronal processes when microwave heating was used instead of processing at room temperature. For this purpose, a control group of sections stained according to the conventional method at room temperature was compared to an experimental group stained in a microwave oven at 180 W for 2, 4 and 1 min in 2% silver nitrate, ammoniacal silver nitrate and gold chloride, respectively. Light microscopic examination demonstrated that the normal structure was preserved in both groups and that there was no difference in the staining quality between the control and the microwave groups. In addition, staining time for this procedure was reduced to 8 min by using the microwave oven. Our study revealed that microwave irradiation can be used safely for Bielschowsky silver impregnation of neuronal tissues.     

Rapid Bielschowsky silver impregnation method using microwave heating.

The Bielschowsky silver impregnation method has been used extensively to demonstrate neuronal processes including dendrites, axons and neurofibrils. In this study, we examined the differences in the time required for and the staining quality of the Bielschowsky method for neuronal processes when microwave heating was used instead of processing at room temperature. For this purpose, a control group of sections stained according to the conventional method at room temperature was compared to an experimental group stained in a microwave oven at 180 W for 2, 4 and 1 min in 2% silver nitrate, ammoniacal silver nitrate and gold chloride, respectively. Light microscopic examination demonstrated that the normal structure was preserved in both groups and that there was no difference in the staining quality between the control and the microwave groups. In addition, staining time for this procedure was reduced to 8 min by using the microwave oven. Our study revealed that microwave irradiation can be used safely for Bielschowsky silver impregnation of neuronal tissues.     

Enhanced Reliability in Silver Impregnation of Terminal Axonal Degeneration-Original Nauta Method

Brains of rat with surgical lesions 3-5 days old are fixed in 10% neutralized formalin (excess of CaCO₃), 20 μ serial frozen sections cut therefrom and kept in neutralized formalin for an additional 24-48 hr. The sections are soaked in distilled water 12-24 hr, transferred to 50% alcohol containing 0.75 ml of concentrated NH₄OH (sp. gr. 0.91) per 100 ml 12-24 hr, placed in distilled water 2-3 hr and then in silver-pyridine solution (AgNO₃ 3% aq., 20 ml; pyridine, 1 ml) for 48 hr. Test sections are transferred directly to each one of 3 ammoniated silver-solutions, pH 12.8, 13.0 and 13.2, made as follows: To 200 ml of solution 1 (silver nitrate, 6.4 gm; alcohol 96%, 220 ml; NH₄OH (sp. gr. 0.91), 28 ml and distilled water, 440 ml) is added respectively 8-12 ml, 12-16 ml and 16-20 ml of solution 2 (2% NaOH) to give the pH desired. The test sections are studied and the optimal ammoniated silver solution chosen. Two baths of ammoniated silver are used, the section placed with continuous agitation into the first bath for 30 sec and the second bath for 60 sec. The sections are then transferred directly into a reducing bath (formalin 10%, 2ml; alcohol 96%, 5 ml; citric acid 1%, 1.5 ml and distilled water, 4.5 ml) for 2 min and from there to 5% Na₂S₂O₃ for 1 min, rinsed in 3 changes of distilled water, dehydrated and mounted.     

A Modified Pyridine-Silver Stain for Teased Preparations of Motor and Sensory Nerve Endings in Skeletal Muscle

This technique has been developed especially to stain sensory receptors which have been localised intramuscularly by electrophysiological means. Rat intertransverse caudal muscles, removed immediately after death, are fixed for 24 hr in a freshly prepared mixture of absolute ethyl alcohol, 4.5 ml; distilled water, 5 ml; and concentrated HNO_a, 0.1 ml. After a further 24 hr in 10 ml of absolute ethyl alcohol containing 0.1 ml of ammonia solution (sp. gr. 0.88), the muscles are washed in distilled water for 30 min and placed in full strength pyridine for 2 days. They are then washed for 24 hr in distilled water (changed 5-8 times) and left in 2% AgNO₃, in the dark for 3 days at 25 C. Following reduction in 10 ml of 5% formic acid containing 0.4 gm of pyrogallol for 6-24 hr, the specimens are washed briefly in distilled water and stored in pure glycerol. The nerve endings can then be teased out and mounted in glycerol, under cover glasses ringed with a waterproof cement. The advantage of this method is that it gives consistently good staining of receptors and motor end-plates in small muscles of the rat     

Osteopontin is histochemically detected by the AgNOR acid-silver staining

Silver nitrate staining of decalcified bone sections is known to reveal osteocyte canaliculi and cement lines. Nucleolar Organising Regions (NOR) are part of the nucleolus, containing argyrophilic proteins (nucleoclin/C23, nucleophosmin/B23) that can be identified by silver staining at low pH. The aim of this study was to clarify the mechanism explaining why AgNOR staining also reveals osteocyte canaliculi. Human bone and kidney sections were processed for silver staining at light and electron microscopy with a modified method used to identify AgNOR. Sections were processed in parallel for immunohistochemistry with an antibody direct against osteopontin. Protein extraction was done in the renal cortex and decalcified bone and the proteins were separated by western blotting. Purified hOPN was also used as a control. Proteins were electro-transferred on polyvinylidene difluoride membranes and stained for AgNOR proteins. In bone, Ag staining identified AgNOR in cell nuclei, as well as in osteocyte canaliculi, cement and resting lines. In the distal convoluted tubules of the kidney, silver deposits were also observed in cytoplasmic granules on the apical side of the cells. Immunolocalization of osteopontin closely matched with all these locations in bone and kidney. Ag staining of membranes at low pH revealed bands for NOR proteins and 56 KDa (kidney), 60KDa (purified hOPN) and 75 KDa (bone) bands that corresponded to osteopontin. NOR proteins and osteopontin are proteins containing aspartic acid rich regions that can bind Ag. Staining protocols using silver nitrate at low pH can identify these proteins on histological sections or membranes.     

Wool Fibres: Sectioning and Staining, Differentiation of Ortho and Paracortex

A double embedding technique for tangential sectioning of hair and wool fibres is as follows: The cleaned fibre bundle is attached to a U-shaped, 16 gauge, tinned-copper wire frame with collodion adhesive, soaked in 6% nitrocellulose for 1 hr, and treated with chloroform for 2 hr. The hardened bundle is then cut fom the wire support and embedded in paraffin-beeswax, 95:5. Sectioning is at 6-8 μ. The use of 2% orange G or saturated aqueous picric acid for quantitative study of the fibres, and the demonstration of wool fibre cortical fractions by staining with polychrome methylene blue after oxidation of the sectioned fibres in a solution of formic acid (98/100 w/v) 25 ml; distilled water, 65 ml; and H₂O₂ (30% w/v), 10 ml, for 1 hr, is recommended.