Straight Oso4 versus Glutaraldehyde-Oso4 In Sequence as Fixatives for the Granular Vesicles in Sympathetic Axons of the Rat Pineal Body

Pineal bodies were removed immediately after death from 6 rats: representing both sexes, and adult and 21-day postnatal ages; cut into 2 or 3 pieces, and subjected to experimental fixations at pH 7.3, 0-4 C as follows: 1-2 hr in 1% OsO₄, with veronal-acetate buffer of phosphate buffer; 3-4 hr in 3% or 6% glutaraldehyde in 0.1 M or 0.2 M phosphate buffer, with or without 1% sucrose. Specimens from OsO₄ were dehydrated, and embedded in epoxy resin; those from glutaraldehyde were allowed to soak in buffer for 12-16 hr, then transferred to 1% OsO₄ at 0-4 C for 2 hr, and embedded in the same manner as the ones fixed directly in OsO₄. Representative electron micrographs of postganglionic sympathetic endings were studied for the morphology and frequency of granular vesicles. No consistent difference was shown between vesicles fixed in OsO₄ buffered by phosphate or by veronal-acetate, nor was there any effect caused by the different concentrations used for the glutaraldehyde solution; however, vesicles fixed by the glutaraldehyde-OsO₄ sequence showed an enhancement in the graininess of their membranes, were slightly larger, and had a much larger dense core than those fixed by OsO₄ alone. After glutaraldehyde-OsO₄, granular vesicles showed a frequency of 81%, whereas after direct fixation in OsO₄, only 40% without significant change their number per unit area. Therefore, glutaraldehyde-OsO₄ seems to be more effective than straight OsO₄ for the demonstration of granular vesicles in the autonomic nervous system.     

Preservation of fine Structures in Yeast by Fixation in a Dimethyl Sulfoxide-Acrolein-Glutaraldehyde Solution

The primary fixative containing 2% acrolein, 2% glutaraldehyde in 50% aqueous dimethyl sulfoxide (DMSO) buffered at pH 7.4, was applied for 7 hr in the cold. After a short wash in 0.02 M s-collidine buffer, pH 7.4, containing 0.2 M sucrose and 0.001 M CaCl₂, the yeast cells were postfixed in 3% OsO₄ at pH 4.0 (veronal acetate buffer). This method preserves many cytoplasmic features such as lipid deposits and ribosomes which are usually destroyed by permanganate fixation. DMSO apparently acts as a permeating agent allowing maximum penetration of the cell wall by the fixative without disrupting cellular fine structure     

The Quantitative Determination of Osmium Tetroxide in Fixatives

This rapid spectrophotometric method for determining the OsO₄ concentration in fixative and stock solutions is based on the reduction of OsO₄ by acidified KI to the blue species of OsI₆ =, which is then determined at 649 mµ. The salt K₂OsI₆ has been isolated from the reaction mixture and characterized. Method: A I ml aliquot of the solution, containing up to 3% OsO₄, is diluted to 100 ml with distilled water. To 1 ml of the diluted solution is added, in order: distilled water, 2 ml; 1 M HCI, 1 ml; and 1 M KI, 1 ml. Optical density at 649 mµ is read from 10-120 min thereafter. OsO₄ concentration is calculated from the measured molecular extinction coefficient of OsI₆ =, 4400 liter/mole cm.     

Maillet's Oso4-ZnI2 Fixative and Alcian Blue Staining in the Study of Neurosecretion; Invertebrates

Maillet's OsO₄-ZnI₂ fixation staining can be combined with a subsequent counterstaining by Alcian blue or aldehyde fuchsin to demonstrate neurosecretory cells in addition to cytological details of the nerve tissue. This technic has been applied to various annelids: Eisenia foetida (Oligochaeta), Erpobdella octoculata (Achaeta) and Nereis diversicolor (Polychaeta). The material is fixed in a 1:4 mixture of 2% OsO₄ and 3% ZnI₂ for 15 nr, embedded in paraffin, sectioned at 5 μ and the sections alternatively mounted on two glass slides. One of these is oxidized by a solution of 0.3% KMnO₄ acidified by 0.6% H₂SO₄ and counterstained with 1% Alcian blue, pH 0.2, the other one is mounted in balsam. The two preparations may then be compared to locate the neurosecretory cells among the other neurons shown on a slide treated only by the OsO₄-ZnI₂. Secretory cells are not stained by Maillet's reagent; except for their Golgi bodies and their cellular and nuclear membranes. The zone of grains which is generally strongly stained by the Alcian blue takes a yellowish hue from the OsO₄-ZnI₂ fixation. This method could be successfully applied to the histological controls in regeneration experiments. In these last ones, we must simultaneously observe the regeneration of the nervous fibres and the possibility of intervention of neurosecretory elements.     

An Oxidation-Distillation Procedure for Reclaiming Osmium Tetroxide from Used Fixative Solutions

A relatively simple method for recovering more than 80% of the osmium from used fixative solutions as OsO₄ is described. The solutions that we have worked with contained excess FeSO₄, which is added routinely to reduce the tetroxide to the dioxide and make them safe for disposal. The solutions also contained other materials such as cacodylic acid (dimethylarsinic acid), mono-sodium phosphate, sodium veronal (a buffer containing barbital and sodium acetate), NaCl, CaCl₂, various sugars, and such lipids as may have been extracted from animal tissue during fixation.     

Effects of Fixation on the Morphology of Paneth Cell Granules

Mouse Paneth cell granules fixed with formalin have been reported to be composed of a core consisting of a polysaccharide-protein complex which is surrounded by a shell or “halo” of acid mucopolysaccharide. Electron micrographs of granules fixed with 1% OsO₄ in veronal-acetate buffer show that the halo may be merely a result of shrinkage of the granule. Similar results obtained after fixation with either 1% OsO₄ in s-collidine buffer containing 1% sucrose (OsO₄) or with 3% glutaraldehyde in cacodylate buffer containing 1% sucrose (GAS) strengthen the belief that the halo is a shrinkage space. Fixation in GAS for 1-4 hr followed by fixation for 1 hr in OsO₄, however, reduced the halo to negligible proportions. This sequence of GAS and OsO₄ improved the preservation of other cytoplasmic structures also. Staining with Alcian blue failed to demonstrate acid mucopolysaccharide in the halo; therefore, the separation of granule components into a core and a halo is probably a fixation artifact.     

Differentiating Type i and Type ii Alveolar Cells in Rat Lung by OsO4NaI Staining

The fixing-staining mixture consisted of 1 part of 2% aqueous OsO₄ and 3 parts of 3% Nal in distilled water. Fresh lungs were cut into 2 mm slices and immersed in this solution for 24 hr at room temperature. Controls were fixed in buffered OsO₄ alone. Selective staining of type II alveolar cells was shown by the OsO_t-NaI mixture but was absent in the controls. No additional staining of the sections was required, and the selectivity was readily observable in either paraffin or Araldite sections by light microscopy and in Araldite sections by electron microscopy     

Observations on the Kinetics of Uranyl Acetate and Phosphotungstic Acid Staining of Chromatin in thin Sections for Electron Microscopy

When thin sections of spermatogenic chromatin are fixed with either glutaraldehyde alone or postfixed with osmium tetroxide (OsO₄) and stained with uranyl acetate (UAc) for increasing times, even after as little as 1 min, stain uptake is proportional to section thickness. Greater UAc uptake is observed in chromatin fixed with gutaraldehyde only, but stain uptake is reduced following a long wash with distilled water to a level similar to that seen with postfixed chromatin. Lead citrate poststaining of chromatin fixed with either glutaraldehyde or postfixed with OsO₄ increases UAc uptake by a factor of about 3.

The staining of thin sections of spermatogenic chromatin with ethanolic phosphotungstic acid (PTA) shows a region where stain uptake is proportional to section thickness followed by a plateau. This staining pattern is seen in chromatin fixed with glutaraldehyde alone or postfixed with OsO₄; similar levels for final PTA uptake are also observed.

An increase in the resin content of embedded chromatin postfixed with OsO₄ is proposed to explain the decrease and increase in the rate of migration of UAc and ethanolic PTA staining solutions, respectively.     

A Comparison of Fixation by Formaldehyde and Glutaraldehyde-Formaldehyde for Combined Light and Electron Microscopy of Axonal Degeneration in the Mammillary Body

Mammillary body (Mb) cell structures were compared following four different procedures for fixation, in order to find one procedure which would be suitable both for silver impregnation methods and electron microscopy. Fixatives compared were: (1) formaldehyde (4% pH 5.5), Mb from a frozen-section; (2) formaldehyde (4% pH 7.2.), Mb from a frozen-section; (3) glutaraldehyde-formaldehyde (1%-4% pH 6.0), Mb from a frozen-section; and (4) glutaraldehyde-formaldehyde (3%-2% pH 7.2), unfrozen Mb. All solutions were perfused into the left ventricle of the heart of anesthetized rats, except for the animal from which the unfrozen tissue was obtained. The Mb was excised from the fresh brain and immediately placed in a fixative solution. After aldehyde fixation, the tissues were further fixed in 1% OsO₄ (pH 7.4), dehydrated, embedded in Epon-Araldite and double-stained with uranyl acetate and lead citrate. Comparisons of mitochondria, myelin, synapses, neurofilaments and synaptic membranes indicated that the perfusion of 4% formaldehyde is suitable for both electron microscopy and silver impregnation, and was preferable to the other fixatives tested.     

Increase in the Calcium Content of Cardiac Tissue after Postfixation with Osmium Tetroxide

The concentration of osmium has been measured by destructive chemical analysis in glutaraldehyde fixed heart tissue postfixed with osmium tetroxide and embedded in epoxy resin. After such treatment, the mean atomic number of the specimen (Z) is close to 10, which permits a quantitative analysis of calcium (Ca) by the continuum method, using Z²/A as a correcting factor (A: atomic weight). Wavelength-dispersive X-ray microanalysis has been used to determine the Ca concentration of frog cardiac tissue fixed in glutaraldehyde and embedded in resin. These measurements have been repeated on tissue postfixed in osmium tetroxide; contrary to expectations, the apparent Ca concentration is much higher in osmium-treated than in nontreated tissue. However, this result is observed with OsO₄ solutions prepared in glass, not with solutions prepared in plastic. It is shown by energy dispersive X-ray analysis of droplets that OsO₄ solutions prepared in glass contain large amounts of calcium, potassium and silicon. Care must be taken in preparing OsO₄ fixatives when the fixed tissues are to be subjected to X-ray microanalysis of such elements as Ca or Si.     

Improved Lipid Visualization with a Modified Osmium Tetroxide Method Using Ultrasonic Treatment and Intensification with Imidazole or Triazole

Formalin fixed autopsy tissue containing lipids were cut into 1-5 nun thick blocks, washed well, then postfixed in 2% OsO₄ in 0.03 M veronal acetate buffer for 30, 60, 90, 120, or 180 min with or without ultrasonic treatment. Tissues exposed to ultrasound for 90 min showed superior penetration of OsO₄ and well preserved histological architecture. Tissues also were immersed for 1 hr in veronal acetate buffer (pH 7.4) containing 0.5% imidazole or triazole and compared with untreated controls. Paraffin sections, 4 μm thick, were examined under a light microscope with an image analyzer. Both intensity and percentage area of osmium blackening were significantly higher in samples immersed in imidazole or triazole than in untreated controls. No difference was observed between imidazole- and triazole-immersed samples. The OsO₄ method, modified by ultrasound treatment and imidazole- or triazole-immersion, can be applied to routine formalin fixed autopsy materials for improved lipid visualization.     

A morphological and immunolabeling study of freeze-substituted Bacteroides forsythus

We used a rapid freezing and freeze-substitution technique without glutaraldehyde and OsO₄ fixation for the electron microscopic immunocytochemical demonstration of the surface structure of Bacteroides forsythus, an anaerobic Gram-negative periodontopathogen. Cells were applied to pieces of filter paper and freeze-substituted by plunge-freezing in liquid propane, substituted in methanol containing 0.5% uranyl acetate, and infiltrated with LR White resin. The membrane ultrastructure of B. forsythus was preserved well, and the labeling density of the freeze-submitted cells was compared to a conventional processing method. Our results show the usefulness of the freeze-substitution method for immunohistochemical studies of B. forsythus.     

Formalin-Chloride Fixation to Improve Silver Impregnation of the Golgi Apparatus

The addition of a 1% concentration of alkaline earth or heavy metal chloride to a 15% neutral formalin solution was found to be much superior to a similar addition of nitrate. Of the 19 salts tried, BaCl₂ and CoCl₂ generally gave the best results with the tissues of Pheretima posthuma, Pila globosa, Rana tigrina, Cauia porcellus and Mus rattus. The CoCl₂ fixative was found to work more successfully with shorter time for fixation and subsequent impregnation by silver, than da Fano's cobalt nitrate technique. The results obtained with BaCl₂ fixative, were equally good and in some cases even excelled those obtained with Aoyama's CdCl₂. Fixatives containing MgCl₂ and ZnCl₂ were satisfactory but the results were not so good as were those obtained with barium and cobalt chloride fixatives. Fixatives containing nitrates generally required longer periods for impregnation and reduction, than those containing chlorides.     

Handling Germinated Pollen on Millipore Membrane During the Feulgen and Autoradiographic Procedures

To prevent loss of pollen during the Feulgen's procedure, the pollen was grown on an autoclaved membrane filter (Millipore AA WP 025 00) in contact with a sterilized medium containing agar 0.5-1%, sucrose according to the genus (Malus 0.3-0.5 M; Persica and Tulipa 0.4 M), and H₃BO₃, 0.01%. To fix the germinated pollen of most species, the membrane was placed for 2 hr to overnight at 2-4 C on filter paper wet with the following mixture: OsO₄, 1 gm; CrO₃, 1.66 gm; and distilled water, 233 ml. To fix Persica pollen, 10% of glacial acetic acid had to be added to the fixative. Washing with distilled water and bleaching with a mixture of 3% H₂O₂ and sat. aq. ammonium oxalate, 1:1, were performed also on filter paper. Similarly, the preparation was processed for Feulgen staining by use of pieces of filter paper wet with the required fluids. Hydrolysis preceding the Schiff's reagent was performed at room temperature with 5 N HCl for 18 min. The differentiation after the Schiff's action was with 2% K₂S₂O₅ buffered to pH 2.3 with 9 ml of phosphate buffer (KH₂PO₄, 1.4 gm; conc. HCl, 0.35 ml and distilled water to make 100 ml). The stained pollen was floated off the membrane with a drop of glacial acetic acid to a gelatinized or an albumenized slide, and squashed. When the coverslip is removed the preparation may be either dehydrated and mounted or coated with autoradiographic film.     

Staining Myelin Sheaths of Optic Nerve Fibers with Osmium Tetroxide Vapor

OsO₄ solution in water, long regarded as the best fixing and staining agent for myelin sheaths, has poor penetrating power. This peculiarity has limited its use to very small pieces of tissue. The vapor from an aqueous solution is known to have a much greater penetrating power for non-neural tissues than the solution itself but nothing has been recorded about its advantages for fixing and staining myelin sheaths of nerve fibers. Difficulties in securing adequate staining of the myelin sheaths in vertebrate optic nerves were overcome largely by the use of the vapor of OsO₄. The technic is carried out as follows: 1) suspend a portion of the nerve above a 2% solution of OsO₄ for 12-24 hours in an air-tight container at room temperature; 2) wash 4-6 hours in distilled water, dehydrate in ethyl alcohol (50% for 2 hours, 70% for 2 hours, and finally 95% overnight), and transfer to n butyl alcohol (2 changes of 2 hours each); 3) embed in paraffin, section, mount and cover in balsam in the customary manner.     

Phosphotungstic Acid-Chromic Acid as a Selective Electron-Dense Stain for Plasma Membranes of Plant Cells

A mixture consisting of 1% phosphotungstic acid (PTA) in 10% chromic acid (CrO₃) selectively stains the plasma membrane of plant cells. Whole tissue or pelleted cell fractions are prepared for electron microscopy using conventional methods including glutaraldehyde fixation and OsO₄ postfixation, dehydration in acetone and embedding in Epon. To stain the plasma membrane, thin sections are transferred with a plastic loop to the surface of a 1% aqueous solution of periodic acid for 30 min for destaining. Following transfer through 5 distilled water rinses, the sections are exposed to the PTA-CrO₃ mixture for 5 min, rinsed and mounted on grids for viewing with the electron microscope. The selectivity of the stain is retained in homogenates and serves to identify the plant plasma membrane in cell fractions.     

Staining Residual Lipids in Ultrathin Sections of Tissues Embedded in Polyester Resin

Rat suprarenal glands fixed in Palade's 1% OsO₄, buffered at pH 7.7 with veronal-acetate, to which 0.1% MgCl₂ was added, were embedded in Vestopal-W and sectioned at 0.2-1 µ. The sections were attached to slides by floating on water, without adhesive, and drying at 60-80° C, placed in acetone for 1 min and then treated with the following staining procedure: Place the preparation in a filtered solution of oil red O, 1 gm; 70% alcohol, 50 ml; and acetone, C.P., 50 ml; for 0.5-1 hr. Rinse in absolute ethyl alcohol; drain; counterstain with 0.5% aqueous thionin for 5 min; rinse in distilled water; drain; stain in 0.2% azure B in phosphate buffer at pH 9, for 5 min. Dry and apply a drop of immersion oil directly on the section. The preparations are temporary. Ciaccio-positive lipids, rendered insoluble by OsO, fixation, stained red to ochre.     

The Use of Dimethylsulfoxide for Fixation of Yeasts for Electron Microscopy

Conventional methods of chemical fixation are often inadequate for preserving yeast ultrastructure. The thick cell wall severely limits penetration of fixatives rendering poor detail of the cell wall, membranes, and overall anatomy. Dimethylsulfoxide (DMSO) enhances penetration of chemicals and has been added to fixatives to improve cell preservation. At high concentrations (5 to 50%), however, it affects ultrastructure unpredictably. We found that adding 0.1% DMSO to fixatives greatly improved retention of yeast ultrastructure. Candida albicans, C. glabrata and Aspergillusfumigatus were fixed for 3 hr in 3% paraformaldehyde, 1% glutaraldehyde, 1 mil MgCl₂, 1 mM CaCl₂, 0.1% DMSO in 0.1 M sodium cacodylate buffer followed by 1% OsO₄, 1% K₂Cr₂O₇, 0.85% NaCl. 0.1% DMSO in the same buffer. Thin epoxy sections were post-stained in uranyl acetate and lead citrate. The multilayered character of the cell wall was distinct and well structured. Addition of ruthenium red or alcian blue to the fixatives further enhanced the outer fibrillar layer. The plasma membrane was contiguous and tightly adjacent to the inner manno-protein layer of the cell wall. The cytoplasm was well preserved and the overall preservation of the yeast ultrastructure was significantly improved.     

A high-activity ATP translocator in mesophyll chloroplasts of Digitaria sanguinalis, a plant having the C-4 dicarboxylic acid pathway of photosynthesis

The effect of exogenous adenine nucleotides on CO₂ fixation and oxygen evolution was studied with mesophyll protoplast extracts of the C₄ plant Digitaria sanguinalis. Exogenous ATP was found to stimulate the rate of pyruvate and pyruvate + oxalacetate induced CO₂ fixation, as well as reverse the inhibition of CO₂ fixation by carbonyl cyanide m-chlorophenyl hydrazone and several electron transport inhibitors. The ATP-dependent stimulation of CO₂ fixation varied from 40 to 70 μmol CO₂ fixed/mg chlorophyll per h, suggesting that ATP was crossing the chloroplast membranes at rates of 80–140 μmol/mg chlorophyll per h, since 2 ATP are required for each CO₂ fixed. Fixation of CO₂ could also be induced in the dark by exogenous ATP, in which case ADP accumulated outside the chloroplasts. This suggests that external ATP is exchanging for internal ADP. In contrast, ADP and AMP were found not to traverse chloroplast membranes, on the basis that neither nucleotide inhibited CO₂ fixation or stimulated oxygen evolution that was limited by available ADP for phosphorylation. Further evidence that ATP can enter the chloroplasts was obtained by direct measurements of the increase in ATP in the chloroplasts due to addition of exogenous ATP in the dark. These studies yielded minimal rates of ATP uptake on the order of 30–40 μmol/mg chlorophyll per h. It is suggested that a membrane translocator exists that specifically transports ATP into the chloroplasts in exchange for ADP. The significance of these findings are considered with respect to the C₄ pathway of photosynthesis.     

Hematoxylin-Eosin Staining of OsO4-Fixed Epon- Embedded Tissue; Prestaining Oxidation by Acidified H2O2

Successful application of hematoxylin-eosin staining to 0.5-1 μ sections of OsO₄-fixed Epon-embedded mammalian tissue is made possible by first treating the sections for approximately 1 min at 25-30 C with 10% H₂O₂ acidified with 0.1 or 0.01 N H₂SO₄ to pH 3.2. Subsequent steps are: washing; drying; Hams hematoxylin at 50 C, 1-2 min; washing; drying; 0.2-0.3% NH₄OH in 70% ethanol, 3-5 sec, drying at 50 C; 5% aqueous eosin for 3 & 45 sec at 25-30 C, washing; drying; clearing in xylene and mounting in resin. The use of acidified H₂O₂ prevents the staining of Epon and permits the characteristic staining picture to be obtained. Sections were attached to glass slides without adhesive and processed horizontally on a rack. Slides should be well drained and blotted before each drying step, to prevent formation of precipitate on the section.