An Application of the Frozen Sectioning Technic for Cutting Serial Sections Thru the Brain

The availability of CO₂ ice makes it practical to cut large blocks of cerebral tissue by the freezing method. If the tissue is first treated with 20-30% ethyl alcohol for sufficient time to secure uniform penetration of the alcohol (about 24 hours), formation of hard ice crystals can be controlled and serial sections 25-100 μ thick can be cut with negligible loss. The alcohol can be added to the fixative used for perfusion, or it can be added at any time later in the firing process, or after fixation is completed. The sections are cemented to the slide and groups of slides are manipulated thru staining processes in glass trays. Ordinary cell and fiber stains give satisfactory results. The method is particularly useful for certain neurophysiological purposes such as defining the location of electrode tracks and lesions and certain types of retrogrades. The Prussian blue test for electrolytically deposited iron can be conveniently applied in conjunction with other stains, to determine the point at which a given action potential response was observed, if steel electrodes are used.     

Cutting Unfixed Frozen Sections for Fluorescence Antibody Studies

A simplified method of section cutting, dispensing with guide attachment on microtome blade, and suitable for serial sections of unfixed frozen tissue of 4μ is described. Essential features are low temperature maintenance (—13°C), critical angle of knife and moistening of slides in cold alcohol or isopentane. This method has been found suitable for fluorescence antibody studies where maintenance of low temperatures is necessary.     

Distortion-Free Mounting of Frozen Sections by Handling on Paper Supports

Frozen sections are cut from the specimen until the level of interest is reached. A strip of paper (bond or similar writing paper) 5 cm long and about 1 cm wider than the specimen is moistened with water, closely applied to the surface of the specimen and frozen onto it. As the section is cut, the end of the paper strip above the knife is grasped and turned backward toward the other end of the strip. The section is then applied to an albumenized glass slide, firmed and thawed by finger pressure, and the paper removed. After thorough drying, the preparation is ready for further processing. When properly performed, mounted sections whose details coincide to those of the uncut block can be obtained. If thawing on the knife is prevented by cooling the knife, the technic can be performed without a cryostat, but it is also feasible to use a cryostat if a favorable temperature is maintained. The authors obtained 30 μ serial sections, suitable for stereotaxic mapping, from rabbit brain by this method.     

Paraffin-Beeswax-Stearic Acid: An Embedding Mass for thin Sections

It is possible to cut 1-3 μ sections of rat tissue after passing it through ethanol and chloroform and infiltrating in a wax mixture consisting of 95% Shell Chemical Co. paraffin (MP 125-130° F) and 5% commercial beeswax (clarified by boiling with water and decanting), to which is finally added 10% of technical stearic acid (melted, and clarified by filtration). A potential disadvantage is the slow expansion of the section on the water flotation bath, due to a surface spreading effect of the contained stearic acid. This expansion can be minimised as follows: by adding 0.5% concentration of a secondary alkyl-aryl sulfonate detergent, such as Shell Chemical Co. Teepol, to the notation water; by keeping the temperature of the water at 45° C; by making sure that no section is left on the water for more than 30 sec; and by drying on chemically cleaned slides for 4-18 hr at 45° C., controlled to ±2°. The spreading effect is advantageous in reticulo-endothelial studies, where overlap of cells needs to be reduced to a minimum, and thinly layered cytoplasm expanded.     

A Quick Preparative Method for Electron Microscopy Observations of Delicate Objects Using Alginate Embedding Medium

A quick, safe method has been devised for embedding small or fragile specimens and keeping delicate structures intact. Cells or organisms to be embedded are placed in a viscous sodium alginate solution (1-2%), which is then polymerized in 100 mM calcium chloride. The resulting gel is easily dehydrated, embedded in resin and sectioned for electron microscopy. This method, the alginate gel portion of which was originally developed for the immobilization of Euglena, allows direct observation of each element of the specimens in micrographs. If desired, the alginate can be removed after sectioning by sequestration of calcium in a 20 mM solution of sodium citrate or a 10 mM solution of EGTA. Cells and organelles in the sections respond normally to standard staining procedures.     

Preparation of Thin Sections for Fourier Transform-Infrared Microscopy: a New Embedding Medium for Cellulosic Samples

Fourier Transform-Infrared [FT-IR] microscopy is a combination of instrumentation from which information can be derived about the structure and composition of materials; however, it presents unique problems for sample preparation. Traditional methods of preparing fiber cross sections employ embedding media such as methacrylates, epoxides and polyvinyl alcohols, all of which have groups in common with the cellulose molecule, and absorb in the same regions of the IR spectrum. Therefore, a new embedding method employing polystyrene has been developed for the preparation of cross and longitudinal sections of cellulosic fibers. Although polystyrene is a strong IR absorbing material, it can be completely removed from specimens prior to analysis. In addition, FT-IR spectra of cross sections have better resolution than conventional preparation methods employing ground samples prepared in a KBr disk.     

An Edible-to-insects Calcium Alginate Gel as a Carrier for Entomopathogenic Nematodes

A carrier for entomopathogenic nematodes based on an edible-to-insects calcium alginate gel was developed. The alginate system was produced by external setting through an interaction between an aqueous sodium alginate mixture and calcium ions under acidic conditions. Sodium hexa-metaphosphate was used to control gel formation. Yeast extract used in the gel as a phagostimulant for Spodoptera littoralis larvae improved the insect's relative consumption rate and digestibility. The nematodes in the gel effectively controlled the larvae in a 24-h leaf bioassay, although nematode survival in the gel was ~ 50%. Gels subjected to 31% relative humidity (RH) prior to larval feeding became desiccated and were inedible to insects. However, gels at 61% RH supported larval feeding, although the water loss from the gel due to evaporation from 200-400-mg gel cubes at this humidity exceeded 50%. The gel might be a useful delivery system for nematodes against insects infesting the plant canopy in greenhouses.     

An Adhesive Transfer Method of Cutting and Mounting thin Sections for Autoradiography

A short strip of adhesive transfer cellophane tape is applied to the face of the block to provide backing for the section during cutting and handling. The tape is sealed to the nuclear track plate with the section against the dry emulsion. After exposure the cellophane backing is removed by immersion in water, and the adhesive is dissolved from the section in unleaded gasoline. The section is hydrated and photographic and histological processing are carried out in the usual manner.     

A Durable Nissl Stain for Frozen and Paraffin Sections

Frozen sections, 25-50 /j. thick, of formalin-fixed nervous tissues are mounted following the Albrecht gelatin technic. Paraffin sections, 15 p., are deparaffinized and transferred to absolute ethanol. The slides are then coated with celloidin. Both frozen and paraffin sections subsequently follow the same steps: absolute ethanol-chloroform (equal parts) for at least 20 min, 95% ethanol, 70% ethanol (1-3 min), then rinsed in distilled water. Sections are stained in Cresylechtviolett (Chroma) 0.5% aqueous solution containing 4 drops of glacial acetic acid per 100 ml, rinsed in distilled water, agitated in 70% ethanol until excess stain leaves the slide, and rinsed in 95% ethanol. Sections are then dehydrated in absolute ethanol, followed by butanol, cleared in xylene, and enclosed in permount.     

Staining and Washing Ultrathin Sections; a Device for Handling Various Materials Simultaneously

To eliminate individual manipulation, as many as 10 grids, each held firmly by a small notched bar of polyethylene plastic, are simultaneously stained, then washed. If the stain used is reactive with atmospheric CO₂ it can be forced through a Millipore filter into a small chamber made of glass tubing which contains the grid holder. The stain, cleared of any solid particles, has very little contact with air and remains free of lead carbonate contamination. Washing is carried out by submerging the chamber and removing the grid holder under water (Feldman, D. G., J. Cell Biol., 15: 592-5, 1962). Washing is minimized because there is not the risk of contaminating grids and wash water with stain trapped between the points of forceps. The polyethylene is nonadherent to the wash water, and the grids can therefore be dried quickly on the holder. With this method, the relative stainability of different materials may be observed because each grid within a batch receives identical treatment.     

Rapid Nissl Staining for Frozen Sections of Fresh Brain

Working with X-ray film autoradiography of soluble isotopes, we needed a staining technique for the localization of nuclei in frozen sections of fresh brain. We have found no Nissl staining method in the literature concerning autoradiography specially recommended for this purpose, nor have we found in handbooks on staining a Nissl method clearly recommended for unfixed, frozen sections of brain. The methods described are intended for paraffin or celloidin sections, and require fixation of brain before sectioning (which must be avoided when working with soluble isotopes). Because autoradiography is a time-consuming method, any technique which shortens time needed for the overall procedure is welcome. Most Nissl techniques described in the literature require long preprocessing of the tissue. We found two rapid methods, described by Humason (1967) and LaBossiere and Glickstein (1976), but their application to frozen sections did not give good results. After trials with several types of techniques, we succeeded in developing two Nissl modifications with slightly different qualities, one of 12 min and the other of 2-3 h. The longer method includes conventional steps in staining; the shorter method does not include fixation or lipid extraction. These methods were applied to 20-60 μm brain sections cut in the cryostat at -10 to -12 C and dried on gelatinized slides.     

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles ¹ and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies ². Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.     

Staining Pericellular Boutons of Glutaraldehyde-Fixed Nervous Tissue; A Chromation-Silvering Sequence for Frozen Sections

After fixing in phosphate-buffered 5% glutaraldehyde, pH 6.8, by perfusion, brains were sliced to 3-5 mm pieces which were placed in the fixative for 5-7 days. The pieces were washed through several changes of 2.26% NaH₂PO₄ for 12 hr, 30 μ frozen sections cut, and mordanted 2 days in an equal-parts mixture of 3.5% CrO₃ and 5% Na-tartrate, which had been aged at 20-25 C for 20 days prior to use. After washing in distilled water, the sections were put into a solution containing AgNO₃, 20 gm; and KNO₃, 15 gm, in distilled water, 80 ml; at 30 C for 1.5-2 hr, then reduced at 40-45 C in three pyrogallol solutions as follows: 1-2 sec in 1% pyrogallol in 55% alcohol; 3-4 sec in a 0.67% solution in 33% alcohol, and 5-7 sec in a 0.5% solution in 25% alcohol. Gold toning is optional; dehydration, clearing and covering, routine. The technic shows particularly the perisomatic fibers, boutons en passant and boutons termineaux. Fibers in nerve tracts may be visible but lightly stained; cell nuclei may be dark, but the cytoplasm remains pale.     

Open-Face, Epoxy Embedding of Single Cells for Ultrathin Sections

Intact stamens of Tradescantia were fixed, dehydrated, and infiltrated with an epoxy resin. Each stamen was then put into a drop of resin on a microscope slide, which was transferred to the stage of a dissecting microscope so that individual hairs could be detached from the filament with fine tungsten needles. The detached hairs were transferred to drops of resin ca. 2 mm in diameter (6 or 7 in each of two rows) lying on a slide heavily coated with evaporated carbon. Polymerization was carried out in an oven until the resin attained a degree of viscosity that permitted orientation of the isolated hairs (by using a compound microscope) without their subsequent dislocation. When the small drops of resin had hardened after further polymerization, the positions of the hairs were marked by circumscribing the cells with India ink. The block was pried from the slide after rapid cooling with solid CO₂, and was then trimmed and sectioned. Cells suspended in culture medium were embedded in much the same way; they were centrifuged to obtain a pellet, which was fixed, dehydrated, and infiltrated. A small fragment of the pellet with a little resin was placed on a microscope slide, where the cells were dissociated under a dissecting microscope at ca. 100 × magnification. Individual cells were then picked up with tungsten needles and transferred to droplets of resin on a carbon-coated slide. The subsequent steps were similar to those described for the staminate hairs. Pieces of tissue in the 50-500 μ range were also handled by the foregoing technique. However, after infiltration they were put into large drops of resin on a slide coated with silicone mold-release rather than on a surface coated with carbon.     

An Inexpensive Microtome Attachment for Cutting 50-Micron Nonfrozen Sections for Electron Microscopic Histochemistry

The attachment is for an American Optical Co., Model 888 freezing microtome. It consists of right-angle bracket made from % inch metal rod, one end of which M clamped in the freezing stage holder; the other supports a tissue holder whose base lies at a right angle to the usual position. The fixed and blotted tissue specimen is enclosed (without infiltration) in parah, on a piece of filter paper which is attached to the base of the tissue holder, and sectioned across its long axis by 50 μ increments. After sectioning, the filter paper is removed from the base, the paraffin matrix opened, and the sections transferred to the appropriate processing fluid     

Polystyrene Embedding and Spreading of Sections at Lower Temperature

Embedments of histological material for the light microscope using a polystyrene solution (Frangioni and Borgioli Polystyrene Embedding Medium, Polysciences Inc., Catalog no. 15858) has several advantages (Frangioni and Borgioli 1979) including: ease of use, consistency of results, possibility of obtaining sections of any thickness using even a steel knife microtome, possibility of using all staining methods applicable to paraffin embedments, and superior quality of the mounts. Excellent preservation of morphology has been confirmed by pictures taken under the electron microscope of osmium fixed material embedded in polystyrene (Frangioni and Borgioli 1979).     

Polystyrene Embedding and Spreading of Sections at Lower Temperature

Embedments of histological material for the light microscope using a polystyrene solution (Frangioni and Borgioli Polystyrene Embedding Medium, Polysciences Inc., Catalog no. 15858) has several advantages (Frangioni and Borgioli 1979) including: ease of use, consistency of results, possibility of obtaining sections of any thickness using even a steel knife microtome, possibility of using all staining methods applicable to paraffin embedments, and superior quality of the mounts. Excellent preservation of morphology has been confirmed by pictures taken under the electron microscope of osmium fixed material embedded in polystyrene (Frangioni and Borgioli 1979).     

Some Improvements in the Preparation of Fresh Frozen Sections

For the histochemical demonstration of sensitive enzymes it is necessary to use fresh unfixed tissue sections. With the following procedure one can constantly obtain such sections 10-20μ thick with relative ease. Schanze's sliding microtome is employed. The microtome knife is deeply cooled by placing blocks of dry ice on its surface, and is provided with a device for preventing the sections from rolling up. The microtome is operated in an ordinary refrigerator maintained at a temperature of 0-3°C. For this purpose, the door of the refrigerator is replaced by a wooden door provided with a glass window, gloved arm holes, and a small door.