Use of a Tissue Sectioner to Expose Internal Structures of Biological Samples for Scanning Electron Microscopy

A procedure yielding sections of unembedded biological samples for observation by scanning electron microscopy is described. Sections of samples, fixed and hardened in OsO₄, were obtained in quantity with a tissue sectioner. Subsequent treatments to osmium-coat cut surfaces were employed prior to critical point drying. The procedure yields cleanly cut surfaces through cells and cytoplasmic organelles which are retained in their normal position. Sections of apple leaf and mouse kidney are illustrated. Sections can be readily cut in a desired plane with less structural damage than is typically encountered by other sectioning or dissection techniques.     

Use of a tissue sectioner to expose internal structures of biological samples for scanning electron microscopy.

A procedure yielding sections of unembedded biological samples for observation by scanning electron microscopy is described. Sections of samples, fixed and hardened in OsO4, were obtained in quantity with a tissue sectioner. Subsequent treatments to osmium-coat cut surfaces were employed prior to critical point drying. The procedure yields cleanly cut surfaces through cells and cytoplasmic organelles which are retained in their normal position. Sections of apple leaf and mouse kidney are illustrated. Sections can be readily cut in a desired plane with less structural damage than is typically encountered by other sectioning or dissection techniques.     

Facilitated ultracytochemical demonstration of retrograde axonal transport of horseradish peroxidase in peripheral nerve

p-Phenylenediamine/pyrocatechol mixture (PPD-PC) was evaluated as a reagent for the ultracytochemical demonstration of retrograde axonal transport of horseradish peroxidase (HRP). HRP crystals were applied to the proximal stumps of the severed infraorbital nerves in rats. After 48 h the rats were sacrificed by perfusion, and the trigeminal ganglia ipsilateral to the severed nerves were processed for HRP cytochemistry and then prepared for electron microscopy. PPD-PC was rapidly oxidized in HRP-labeled neurons to form a dark brown-black osmiophilic reaction product which was more readily visible than the DAB product in the sections. This facilitated selection by light microscopy of areas in the epoxy wafers for ultrathin sectioning. In thin sections viewed under the electron microscope, the osmicated electron opaque PPD-PC reaction product was present in membrane-bound structures including smooth endoplasmic reticulum and granules of various sizes. The PPD-PC reaction product formed after 10-min incubation appeared to be more electron opaque than the DAB reaction product formed after 20 min. PPD-PC was found to be much less readily oxidized than DAB by endogenous hemoproteins. This methodology facilitated the ultracytochemical localization of HRP in neurons following retrograde axonal transport.     

Sections from double mesas obtained at the same tissue plane

A method for obtaining sections from two areas in the face plane of a tissue block is described. It facilitates ultrathin sectioning where virtually identical planes of section are essential but where areas of interest are too far apart to be included in a single section. Two horizontally separated mesas are prepared; sections are cut from the first with the knife rotated around its vertical axis by 2-3 degrees to provide clearance for the other. The second mesa is then sectioned with the knife rotated 4-6 degrees in the opposite direction. Similarly, by changing the vertical inclination of the block, two additional vertically separated mesas can be cut. This procedure is of great value for comparative morphometric studies of material from opposite sides of individual specimens.     

Sections from Double Mesas Obtained at the Same Tissue Plane

A method for obtaining sections from two areas in the face plane of a tissue block is described. It facilitates ultrathin sectioning where virtually identical planes of section are essential but where areas of interest are too far apart to be included in a single section. Two horizontally separated mesas are prepared; sections are cut from the first with the knife rotated around its vertical axis by 2-3° to provide clearance for the other. The second mesa is then sectioned with the knife rotated 4-6° in the opposite direction. Similarly, by changing the vertical inclination of the block, two additional vertically separated mesas can be cut. This procedure is of great value for comparative morphometric studies of material from opposite sides of individual specimens.     

Immunohistochemical investigation on the presence of chondroitin sulfate in calcification nodules of epiphyseal cartilage.

Chondroitin sulfate localization in mouse epiphyseal cartilage was studied using CS-56 monoclonal antibody immunospecific for the glycosaminoglycan portion of the molecule. For light and fluorescence microscopy, decalcified specimens were embedded in paraffin, Lowicryl, or were frozen and cryostat-sectioned, and the antigen-antibody reaction was demonstrated by treating sections with IgM-peroxidase, IgM-alkaline phosphatase, or IgM-fluorescein conjugates. For electron microscopy, decalcified and undecalcified specimens were embedded in Lowicryl; ultrathin sections from undecalcified specimens were decalcified by flotation on EDTA; sections from both types of specimens were treated with IgM-immunogold conjugate for demonstration of CS-56 reaction. Before immunoreaction, part of all decalcified sections were digested with Streptomyces or testicular hyaluronidase. Control sections were treated with either mouse and goat non-immune serum, or mouse monoclonal antiserum to human dendritic reticulum cells. Both light and electron microscopy show CS-56 reaction with cytoplasmic components of maturing and hypertrophic chondrocytes. Under the light microscope, immunoreaction was not visible in calcified matrix, and was visible in uncalcified matrix only after hyaluronidase digestion. Under the electron microscope, it was evident both in uncalcified and calcified matrix, although the latter showed few immunogold particles, usually placed on areas which appeared incompletely calcified. Gold particles were chiefly distributed at the periphery of calcification nodules and fully calcified matrix. These results show that CS-56, besides reacting with cytoplasm of maturing and hypertrophic chondrocytes, binds to crystal ghosts and other components of cartilage matrix, immunoreactivity decreasing as calcification increases. This suggests that chondroitin sulfate molecules are either degraded during calcification, or segregated into macromolecular complexes, or both degraded and segregated. The second possibility is supported by the increase of immunosensitivity induced by hyaluronidase digestion.     

The demonstration of arylsulfatases with 4-nitro-1,2-benzenediol mono(hydrogen sulfate) by the formation of osmium blacks at the sites of copper capture.

A new method is described for the direct cytochemical demonstration of lysosomal arylsulfatases utilizing a synthetic substrate, 4-nitro-1,2-benzenediol mono(hydrogen sulfate), and a copper capture reaction. A small amount of Hatchett's brown (cupric ferrocyanide, Cu2Fe(CN)6-7 H2O) formed at the subcellular sites of copper capture is then utilized as a heterogeneous catalyst to effect the oxidative polymerization of 3,3'-diaminobenzidine which results in the formation of an insoluble, highly colored osmiophilic indamine polymer at the sites of enzymatic activity. The reaction product even at this stage prior to osmication is highly visible. It is readily seen with a light microscope in 50 mum sections of fixed tissues prepared with a mechanical chopper or in 10 micron cryostat sections treated for arylsulfatase activity. Upon osmication, an electron-opaque osmium black is formed which is much less soluble than the products of either the lead or barium capture reactions currently used for the demonstration of arylsulfatase with the electron microscope. The selection of areas of plastic-embedded tissues for ultrathin sectioning is facilitated by the ready visibility of these osmium black end products on 1-2 mum plastic sections which can be studied with the light microscope. This method gives permanent specimens demonstrating arylsulfatases A or B in lysosomes and autophagic vacuoles. In addition, enzyme activity is seen occasionally in the Golgi region or lamellae of certain cells believed to be elaborating sulfated products. In these instances, it may be demonstrating sulfotransferase activity.     

Improved Methods for Making and Using Glass Knives

We have observed over time that the right side of a glass knife is the optimal cutting edge for microtomy if the counterpiece (heel opposite the edge) is controlled within 1 mm. The right cutting edge has been considered the “saw toothed” side and has not been used for ultrathin sectioning. We have observed that the right cutting edge is sharper and more durable than the left. Light and scanning electron microscopy were used to observe the cutting edge, and transmission electron microscopy was used to examine semithin and ultrathin sections of animal and plant tissues cut by the right and left sides of the cutting edge. The results indicate that the cutting edge becomes sharper and more durable from left to right. Both the quality and efficiency of ultrathin sectioning is improved by using the right cutting edge.     

Photographic Mapping of a Tissue Surface to Locate Fields for Electron Microscopy; Mouse Lung

Precise sampling from whole lobes of mouse lungs fixed in the inflated state and embedded in epoxy resin can be not only feasible but also efficient. A 1 μm section is cut from an embedded lobe with a rotary microtome and a steel knife. This section is stained and photographed, and from it a 35 × enlarged print is prepared. A grid of transparent plastic scored with 35 mm squares, lettered vertically and numbered horizontally, is superimposed over the photograph. The area chosen for electron microscopy thus becomes identifiable by a letter-number designation obtained from the grid. This area is then located by light microscopy on a 2 mm slice taken from the block from which the 1 μm section was cut, by use of oblique illumination and the calibrated mechanical stage of the light microscope. A block of 1.3 mm diameter is removed for electron microscopy from the tissue by a rotatable circular spring-loaded punch screwed into the objective turret of the microscope. The removed cylinder is mounted on a metal stub and ultrathin sections cut from the faced tissue. The method is as equally suitable for the examination of other tissues, particularly when large areas and multiple sampling may be required.     

Diethylene Glycol Distearate Embedding and Ultramicrotome Sectioning for Light Microscopy

Diethylene glycol distearate can be used as an embedding medium for light microscopy. Two infiltration changes of about 6 hr each in the melted wax (melting point 47-52 C) are required before the final embedding which is done in 00 gelatin capsules for sectioning in the ultramicrotome by the procedure used in electron microscopy. Serial sections 1-2 μ thick can be cut without difficulty. No cooling devices are necessary for trimming and sectioning at laboratory temperature. Sections rarely become detached from the slides. The staining characteristics of the tissues are the same as when embedded in paraffin. For fluorescence microscopy, essentially the same procedure is followed. Tissues are not distorted and the intracellular structures are well preserved.     

Light and electron microscopic structure of golgi-stained neurons in the vertebrate brain (new rapid Golgi procedure)

Summary After application of a rapid, selective silver impregnation procedure for light (LM) and electron (EM) microscopy, individual neurons are distinguishable by a light silver precipitation. The silver content is sufficient that entire nerve cells can be observed light microscopically; on the other hand, electron microscopically the cytological details are still visible. Brains of mice were fixed by phosphate-buffered aldehyde perfusion, and pieces of tissue left in a 1 % K₂Cr₂O₇ solution for 13 h before impregnation in a 0.5 % AgNO₃ solution for 2h. Thick sections (30–50 m) of the impregnated tissue were cut; from these sections, suitably stained neurons were dissected out and re-embedded for ultrathin sectioning, thereby allowing observations on the same neurons at the EM level. A thin silver deposit was observed along the delimiting neuronal membrane, the microtubules and the smooth ER, including the spinal apparatus of the dendritic spines. The fine cytoplasmic details of the impregnated neurons and the surrounding tissue are well preserved and, therefore, suitable for subsequent determination of synaptic relationships of the impregnated neurons with the adjacent neuronal elements.     

Histamine content of peritoneal and tissue mast cells of growing rats

Summary p-Phenylenediamine/pyrocatechol mixture (PPD-PC) was evaluated as a reagent for the ultracytochemical demonstration of retrograde axonal transport of horseradish peroxidase (HRP). HRP crystals were applied to the proximal stumps of the severed infraorbital nerves in rats. After 48 h the rats were sacrificed by perfusion, and the trigeminal ganglia ipsilateral to the severed nerves were processed for HRP cytochemistry and then prepared for electron microscopy. PPD-PC was rapidly oxidized in HRP-labeled neurons to form a dark brown-black osmiophilic reaction product which was more readily visible than the DAB product in the sections. This facilitated selection by light microscopy of areas in the epoxy wafers for ultrathin sectioning. In thin sections viewed under the electron microscope, the osmicated electron opaque PPD-PC reaction product was present in membrane-bound structures including smooth endoplasmic reticulum and granules of various sizes. The PPD-PC reaction product formed after 10-min incubation appeared to be more electron opaque than the DAB reaction product formed after 20 min. PPD-PC was found to be much less readily oxidized than DAB by endogenous hemoproteins. This methodology facilitated the ultracytochemical localization of HRP in neurons following retrograde axonal transport.Supported by NIH Grants DE 04730, DE 02668 and DE 00288 from the National Institute for Dental Research, NIH Grant RR 0533 from the Division of Research Facilities and Resources, and a grant to the Neurobiology Program from the Alfred P. Sloan Foundation     

The Processing of Mammalian Haemopoietic Cells in Thin Layer Agar Cultures for Electron Microscopy

Human and mouse haemopoietic cells cultured by the thin layer agar technique have been studied with the electron microscope. To process colonies of haemopoietic cells or individual cells which appeared in these colonies, a special technique had to be developed. The technique presented covers methods of selection, isolation, and sectioning that were devised for this purpose.

Haemopoietic cells are cultured in small plastic Petri dishes containing a culture system with 0.25% agar. Cell colonies and individual cells intended for light as well as for electron microscopic study are examined and selected microscopically with the aid of a numbered grid which is placed under the closed Petri dish.

Cells in the agar gel are fixed with glutaraldehyde which is pipetted directly onto the cultures. In order to facilitate their removal from the medium, the consistency of the agar solution is increased by evaporating liquid with controlled mild warming.

Pieces of agar containing colonies or single cells are cut out with a fine trephine and postfixed in osmium tetroxide. Agar pieces are embedded cell side up in a thin layer of Epon. After polymerization, the Epon-embedded pieces of agar are appropriately oriented at the head of flat embedding molds filled with fresh Epon. After another polymerization procedure, the top of the Epon blocks containing the cells are trimmed to a smooth surface with a glass knife.

The exact distance between the smooth surface of the blocks and the cells is measured by use of the vertical micrometer of a standard light microscope. The Epon layer around the specimen is trimmed away to expose selected cells for subsequent semi-thick and ultrathin sectioning. Sections are stained and examined microscopically.

With minor modifications the technique described also enables the processing of extremely small quantities of biological materials derived from other experiments for both light and electron microscopic observation.     

A Simplified Resectioning Technique

A simple and rapid technique is outlined for 20-40 mUm sectioning of epoxy embedded tissue, removal of areas of interest from the thick sections, resectiening of the selected areas for ultrastructural use. Use of the embeddmg mixture desaibed by Kushida in 1971- makes possible thick sectioning without fracturing. Mounting on glass slides with immersion oil for light microscopic observation facilitates later demouutng for isolation of selected areas. Identification of specific areas of interest is aided by an osmium intensifier stain, p-phenylenediamine. The method is useful for both localizing specific tissue areas and for systematic sampling at precise intervals.     

Cryo-ultramicrotomy after coating of the cut surface of the tissue block with carbon and metals before sectioning.

Working under improvised conditions it is shown that coating of the cut surface of a frozen tissue block with metal and carbon improves, when applied prior to each cutting, the sectioning properties and the handling of the subsequent ultrathin cryosection.     

Cryo-ultramicrotomy after coating of the cut surface of the tissue block with carbon and metals before sectioning

Summary Working under improvised conditions it is shown that coating of the cut surface of a frozen tissue block with metal and carbon improves, when applied prior to each cutting, the sectioning properties and the handling of the subsequent ultrathin cryosection.     

Block-Surface Staining

A method is described by which the tissue exposed on sectioning a specimen embedded in paraffin can be visualized in situ. The fixed specimen is impregnated with lead acetate, dehydrated in dioxane, infiltrated with paraffin and embedded. Tissues exposed on sectioning are developed by applying to the cut surface of the block a solution of potassium sulphide in water. Concentrations of the reagents used and the time intervals for the procedure are dependent upon the size of the specimen and upon the degree of contrast required. The method is described as it was applied to the study of a small human fetus in cross section. Representative photographs are included to show the results obtained.     

Studies on the Character istic of the Structures in the Cortical Cells of Gastrodia clara after Infection of Armillaria mellea

Aided by the techniques of thin and ultrathin sectioning and electron microscopy, the characteristic of structures in the cortical cells of Gastrodia elata was further investigated after infection of Armillaria mellea. It was found that the “hyphal coils”, observed with light microscope, in the cortical cells of G. elata were saccate structures deriv- ed from the cytoplasm of cortical cells and enclosed hyphae. And the cell walls of hyphae were digested in these sacs. Then, these hyphae without cell wall were cut into protoplast fragments in inner-most cortical cells. The results indicated that the cortical cells of G. elata possess digestive function.     

OBSERVATIONS ON FIBRILLOGENESIS IN THE CONNECTIVE TISSUE OF THE CHICK EMBRYO WITH THE AID OF SILVER IMPREGNATION

A technique is described which combines silver impregnation and ultrathin sectioning for the electron microscopic demonstration of fibrils in the connective tissue of the chick embryo. The electron micrographs presented in this paper provide evidence for the specificity and completeness of the silver-impregnation technique. It has been shown that, in this particular tissue after fixation in neutral formalin and at the stage of development represented by our material, the argyrophil fibers are embedded in a material which is continuous with the body of the fibroblasts.     

A simplified procedure for preparation of undecalcified human bone sections

A new type of apparatus for sectioning samples of hard, undecalcified bone is described. Slices of fresh or archeological human bone 4-5 mm thick are dehydrated and then embedded in epoxy resin. The apparatus used to prepare sections from the resulting blocks consists of a low-speed rim-type diamond cut-off wheel and a slowly advancing table carrying the specimen held in a rotating mount. Sections may be cut at a thickness of 80 micron +/- 1%. After cleaning in an ultrasonic bath, these can be mounted on slides for quantitative microscopic examination with transmitted light. Grinding and polishing are not necessary. The results obtained are illustrated.