Preparation of Large Epoxy Sections for Light Microscopy as an Adjunct to Fine-Structure Studies

Tissue blocks 2 × 2 × 0.4 cm were fixed 6-24 hr in phosphate-buffered 5% glutaraldehyde then sliced to 2 × 2 × 0.1 cm and soaked in 0.1 phosphate-buffer (pH 7.3) for at least 12 hr. Fixation was continued for 2 hr in phosphate-buffered 1-2% OsO₄. The slices were dehydrated, infiltrated with Araldite, and embedded in flat-bottomed plastic molds. Sectioning at 1-8 μ with a sliding microtome was facilitated by addition of 10% dibutylphthalate to the standard epoxy mixture. The sections were spread on warm 1% gelatin and attached to glass slides by drying, baking at 60 C, fixing in 10% formalin or 5% glutaraldehyde and baking again. Sections were mordanted in 5% KMnO₄ (5 min), bleached with 5% oxalic acid (5 min) and neutralized in 1% Li₂CO₃ (1 min). Several stains could then be applied: azure B, toluidine blue, azure B-malachite green, Stirling's gentian violet, MacCallum's stain (modified), tribasic stain (modified) and phosphotungstic acid-hematoxylin. Nuclei, mitochondria, specific granules, elastic tissue or collagen were selectively emphasized by appropriate choice of staining procedures, and cytologic detail in 1-3 μ sections was superior to that shown by conventional methods. Selected areas from adjacent 4-8 μ sections could be re-embedded for ultramicrotomy and electron microscopy.     

Selection for Electron Microscopy of Specific Areas in Large Epoxy Tissue Sections

Tissue blocks 2 × 2 × 0.4 cm were fixed 6-24 hr in phosphate-buffered 6% glutaraldehyde then sliced to 2 × 2 × 0.1 cm and rinsed in phosphate buffer for at least 12 hr. Fixation was continued for 2 hr in phosphate-buffered 1-2% OsO₄. The slices were dehydrated, infiltrated with Araldite, and embedded in flat-bottomed plastic molds. Sectioning at 4-8 μ with a sliding microtome was facilitated by addition of 10% dibutylphalate to the standard epoxy mixture. The sections were spread on water and attached to coverslips by drying, then heating to 80 C for 1 min. Staining 2 min with 1-3% KMnO₄ and temporary mounting in glycerol on a slide allowed the desired area for electron microscopy to be selected and marked. This area was then cemented to the facet of a conventional epoxy casting with a drop of epoxy resin (without added dibutylphthalate). After polymerization, the coverslip was removed by quick cooling leaving a flat re-embedded portion of the original section. This portion was viewed by transillumination in a dissecting microscope and trimmed of surplus tissue. Ultrathin sections for electron microscopy were obtained in the usual manner.     

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.     

Epoxy Embedding, Sectioning and Staining of Plant Material for Light Microscopy

By using a formula which gives a relatively soft epoxy embedding medium, it is possible to cut sections of plant material with a sliding microtome equipped with a regular steel knife. Blocks having a cutting face of 10 × 10 mm, giving sections of 4-10 μm, can be used. Tissues are fixed in Karnovsky's fluid, postfixed in 1 or 2% OsO₄, embedded in Spurr's soft epoxy resin, Araldite, or Epon mixtures. 5% KMnO₄, followed by 5% oxalic acid, then neutralized in 1% LiCO₃, are used to mordant the sections. Some of the stains used are Mallory's phosphotungstic acid-hemotoxylin, acid fuchsin and toluidine blue, or toluidine blue. Mounting is done with whichever soft epoxy resin was used in casting the blocks.     

Processing Pollen and Spore Exines for Electron Microscopy

A resin mixture containing Araldite M, 15 ml; Epon 812, 25 ml; dodecenyl succinic anhydride, 55 ml; and dibutyl phthlate, 2 ml, was found to be the optimal embedding resin for both fresh and acetylated pollen exines. Diamond knives greatly facilitated sectioning. Exine fine structure, and stratification patterns in fresh pollen were most clearly revealed by section staining of glutaraldehyde-fixed (2 hr), OsO₄-stained (2 hr) specimens. Acetylated exines (acetic anhydride-H₂SO₄ 9:1; 100 C, 5 min) did not require additional treatment prior to embedding, but section staining of exines so treated greatly enhanced stain differentiation of exine subunits. Successfully used section stains included Reynold's lead hydroxide, Millonig's lead citrate and aqueous KMnO₄. Additional procedures were tried but were found to have serious disadvantages, e. g. exines treated with KMnO₄ before embedding shattered badly during sectioning.     

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.     

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.     

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.     

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.     

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.     

Ruthenium Red Stain Able Surface Layer on Lung Alveolar Cells; Electron Microscopic Interpretation

Luft's ruthenium red (RR) method was applied to lung tissue. Small blocks of mouse lung were fixed for 1 hr with 1.2% glutaraldehyde at 0-4 C, buffered with 0.067 M cacodylate, pH 7.3 and containing RR, 1 mg/ml. Following fixation, lung blocks were immersed in 0.15 M cacodylate for 10 min and postfixed for 3 hr at room temperature with 2% OsO₄ buffered with 0.067 M cacodylate, pH 7.3, and containing RR, 1 mg/ml. Blocks were dehydrated with ethanol, embedded in Araldite, and ultrathin sections treated with uranyl acetate and lead citrate solutions to enhance contrast of cell structures. Electron micrographs revealed an electron-dense layer coating the exposed surfaces of alveolar cells. This layer corresponded in location and appearance to that observed by other investigators who used colloidal iron techniques.     

The Marchi Method

An investigation has been made of the poor penetration obtained by the Marchi method. Use of surface tension depressants or perfusion technics showed no marked improvement. Optimum results cannot be achieved with slices of tissue above 3 mm. in thickness. Another cause of faulty staining is the failure to maintain an adequate strength of OsO₄ in the Marchi mixture. The concentration of this chemical should never be allowed to drop below 0.25%. A chemical method for determining the amount of OsO₄ present in the staining mixture and based upon the Alvarez test is described. This method can also be utilized to reclaim partially exhausted staining solutions. Remarks are included upon the KIO₃ method of Busch.     

Differentiated Staining of Osmium Tetroxide-Fixed, Vestopal-w-Embedded Tissue in thin Sections

Tissues were fixed at 20° C for 1 hr in 1% OsO₄, buffered at pH 7.4 with veronal-acetate (Palade's fixative), soaked 5 min in the same buffer without OsO₄, then dehydrated in buffer-acetone mixtures of 30, 50, 75 and 90% acetone content, and finally in anhydrous acetone. Infiltration was accomplished through Vestopal-W-acetone mixtures of 1:3, 1:1, 3:1 to undiluted Vestopal. After polymerisation at 60° C for 24 hr, 1-2 μ sections were cut, dried on slides without adhesive, and stained by any of the following methods. (1) Mayer's acid hemalum: Flood the slides with the staining solution and allow to stand at 20°C for 2-3 hr while the water of the solution evaporates; wash in distilled water, 2 min; differentiate in 1% HCl; rinse 1-2 sec in 10% NH,OH. (2) Iron-trioxyhematein (of Hansen): Apply the staining solution as in method 1; wash 3-5 min in 5% acetic acid; restain for 1-12 hr by flooding with a mixture consisting of staining solution, 2 parts, and 1 part of a 1:1 mixture of 2% acetic acid and 2% H₂SO₄ (observe under microscope for staining intensity); wash 2 min in distilled water and 1 hr in tap water. (3) Iron-hematoxylin (Heidenhain): Mordant 6 hr in 2.5% iron-alum solution; wash 1 min in distilled water; stain in 1% or 0.5% ripened hematoxylin for 3-12 br; differentiate 8 min in 2.5%, and 15 min in 1% iron-alum solution; wash 1 hr in tap water. (4) Aceto-carmine (Schneider): Stain 12-24 hr; wash 0.5-1.0 min in distilled water. (5) Picrofuchsin: Stain 24-48 hr in 1% acid fuchsin dissolved in saturated aqueous picric acid; differentiate for only 1-2 sec in 96% ethanol. (6) Modified Giemsa: Mix 640 ml of a solution of 9.08 gm KH₂PO₄ in 1000 ml of distilled water and 360 ml of a solution of 11.88 gm Na₂HPO₄-2H₂O in 1000 ml of distilled water. Soak sections in this buffer, 12 hr. Dissolve 1.0 gm of azur I in 125 ml of boiling distilled water; add 0.5 gm of methylene blue; filter and add hot distilled water until a volume of 250 ml is reached (solution “AM”). Dissolve 1.5 gm of eosin, yellowish, in 250 ml of hot distilled water; filter (solution “E”). Mix 1.5 ml of “AM” in 100 ml of buffer with 3 ml of “E” in 100 ml of buffer. Stain 12-24 hr. Differentiate 3 sec in 25 ml methyl benzoate in 75 ml dioxane; 3 sec in 35 ml methyl benzoate in 65 ml acetone; 3 sec in 30 ml acetone in 70 ml methyl benzoate; and 3 sec in 5 ml acetone in 95 ml methyl benzoate. Dehydrated sections may be covered in a neutral synthetic resin (Caedax was used).     

Preparation of Fossil Bone Specimens for Scanning Electron Microscopy

A method is described for preparing fossil bone specimens for scanning electron microscopy. To obtain bone surfaces suitable for study, material was embedded in Epon 812 and selected faces exposed by grinding were subjected to controlled etching with a 4:1 mixture of 5% HNO₃ and 1% OsO₄, Surfaces thus prepared were further processed by the so-called clearing replicas technique. As a result of this procedure the bone surfaces revealed a network of anastomosing vascular canals the inner surface of whose walls could be examined in the scanning electron microscope. By etching extremely thin ground sections of bone stuck to plastic tape the contents of vascular canals as well as osteocytes can be isolated. This method ensures the good preservation of spatial relations between bone elements essential for studies of fossil bones, which an sometimes very brittle.     

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.     

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 Tribasic Stain for Thin Sections of Plastic-Embedded, OsO4-Fixed Tissues

Thin (0.5-1 μ) sections of plastic-embedded, OsO₄-fixed tissues were attached to glass slides by heating to 70 C for 1 min. A saturated solution combining toluidine blue and malachite green was prepared in ethanol (8% of each dye) or water (4% of each dye). Methacrylate or epoxy sections were stained in the ethanol solution for 2-5 min. The water solution was more effective for some epoxy sections (10-80 min). Epoxy sections could be mordanted by 2% KMnO₄, in acetone (1 min) before use of the aqueous dye, reducing staining time to 5-10 min and improving contrast. Aqueous basic fuchsin (4%) was used as the counter-stain in all cases; staining time varied from 1-30 min depending upon the embedding medium and desired effects, methacrylate sections requiring the least time. In the completed stain, nuclei were blue to violet; erythrocytes and mitochondria, green; collagen and elastic tissue, magenta; and much and cartilage, bright cherry red. Sections were coated with an acrylic resin spray and examined or photographed with an oil-immersion lens.     

Intensification of Osmium Staining by p-Phenylenediamine: Paraffin and Epon Embedding; Lipid Granules in Renal Medulla

Tissue which had been fixed in 4% glutaraldehyde and postfixed in 2% OsO₄ was subsequently treated with p-phenylenediamine, either in the block prior to embedding in paraffin or Epon or, in the case of Epon-embedded material, after sectioning for light microscopy. The p-phenylenediamine was best used as a 0.8-1% solution in 70% alcohol. The p-phenylenediamine caused a very considerable intensification of staining of any cell components; this intensification of staining was particularly marked in the case of the lipid granules of renal medulla.     

Improved Intracellular Morphology of Pneumocystis Carinii From Rat Lung by Postfixation with a Mixture of Potassium Ferrocyanide and Osmium Tetroxide

Pneumocystis carinii infected rat lungs were postfixed with a mixture of OsO₄ and K₄Fe(CN)₆. A marked improvement in staining of cell membranes, endoplasmic reticulum, nuclear membranes and glycogen was observed. These improvements were seen in both the trophic and cystic forms of the organisms. The addition of K₄Fe(CN)₆ did not improve the staining of cell walls, microtubules or ribosomes. Trophozoites were seen attached to both type 1 and type 2 pneumocytes by filopodia and/or intercalation of the cell body of P. carinii with the host lung cells. It is expected that the improvement in ultrastructural detail will allow better understanding of the ultrastructure of P. carinii and provide insights into the modes of action of various antimicrobial compounds on this organism.     

Isolation and Embedding of Single Nerve Cells for Electron Microscopy

Individual neurons have been isolated by freehand dissection under binocular, 40-100 power magnification from the lateral vestibular nucleus of fresh rabbit brain. The cells were fixed with glutaraldehyde and OsO₄ and dehydrated in 33%, 50%, 60% (v/v) and pure Durcupan A prior to embedding in Araldite 502. Blocks were cast in lids of BEEM capsules. The locating of the individually embedded cells and trimming of the blocks were facilitated by means of a holder that permitted visualization in both diffuse transmitted and incident light.