Contributions to Semithin Sextioning on A Conventional Rotary Microtome

Procedures for obtaining sections 1 μ thick on a conventional rotary microtome are described. Hydrophilic resin blocks with adequate hardness and elasticity for semithin sectioning are made by addition of divinylbenzene and methylmethacrylate to a commercial embedding kit. The blocks are pinched between two simple adapters and mounted in the specimen bolder of a microtome. A glass knife of the Ralph type with an effective blade length of 25 mm is made from a glass slide and attached to a metal bar with paraffin. The low cost assembly is set in the steel knife holder of a conventional rotary microtome. Sections I micron in thickness can be cut from the resin embedded blocks. Staining with the usual staining solutions may be weak due to the thinness of the sections, but the fine resolution and low distortion achieved are compensating gains.     

Paraffin Compression Due to the Rotary Microtome

The extent of compression of microtome sections has been studied for blocks with tissue and also blocks of clear paraffin. Thick sections are commonly compressed 15% or more, while in sections below 5 or 10 μ, compression may exceed 50%. Compensatory thickening of sections occurs. The degree of compression for various paraffin samples and for various conditions of knife edge, temperature, etc., is compared. Microscopical work, particularly where quantitative data or reconstructions are involved, is often seriously unpaired by unrecognized artifacts of sectioning. The present work indicates the magnitude of such artifacts. Compensation for distortions of sections is not easy because tissues, particularly dense tissues, may compress less than the paraffin matrix. Section corrugation is due to this inequality in compression. Absorption of water in section flattening causes some tissue readjustment, but this varies with different tissues and different fixations.     

Glass Knife Adapter for Cutting Epoxy Sections on a Conventional Rotary Microtome

Electron microscopy-style fixation followed by epoxy plastic embedding is often now the method of choice for preparing tissue even for light microscopy; I have found it excellent for fluorescence, autoradiographic and conventional histology (Shaw 1972, 1977). Sections more than about five microns thick can be cut on a really sharp steel knife if the plastic is reasonably soft (Stretton and Kravitz 1973, Shaw 1972), but this is much easier and knife marks are reduced if extra-wide glass knives are used on a special-purpose intermediate microtome like the Sorvall JB-4. Recent budgetary restrictions made us defer purchase of such a microtome, and some alternative had to be devised. I report here a simple but rugged adapter for glass knives which replaces the steel knife in a conventional Leitz rotary microtome and allows thin plastic sections to be cut as easily as with a more sophisticated cutter. It could be adapted for any rotary microtome, and can be readily constructed in most machine shops for negligible cost.     

Rapid Dehydration for Celloidin Embedding by Means of Azeotropic Distiliation

By including plant material in a fractional distillation apparatus containing 95% alcohol and benzene it is possible to eliminate water completely in a constant boiling mixture. The dehydrated material can then be embedded in celloidin by orthodox techniques. In spite of the rapidity of the dehydration quite soft tissue as well as woody material can be processed. The method is cheap because it does not require absolute alcohol and is also suited to the humid climate in which it was developed.     

A Protargol Method for Staining Nerve Fibers in Frozen or Celloidin Sections

Extensive experimentation with protargol staining of neurons in celloidin and frozen sections of organs has resulted in the following technic: Fix tissue in 10% aqueous formalin. Cut celloidin sections IS to 25 μ, frozen sections 25 to 40 μ. Place sections for 24 hours in 50% alcohol to which 1% by volume of NH₄OH has been added. Transfer the sections directly into a 1% aqueous solution of protargol, containing 0.2 to 0.3 g. of electrolytic copper foil which has been coated with a 0.5% solution of celloidin, and allow to stand for 6 to 8 hours at 37° C. Caution: In this and the succeeding step the sections must not be allowed to come in contact with the copper. From aqueous protargol, place the sections for 24 to 48 hours at 37° C. directly into a pyridinated solution of alcoholic protargol (1.0% aqueous solution protargol, 50 ml.; 95% alcohol, 50 ml.; pyridine, 0.5 to 2.0 ml.), containing 0.2 to 0.3 g. of coated copper. Rinse briefly in 50% alcohol and reduce 10 min. in an alkaline hydroquinone reducer (H₃BO₃, 1.4 g.; Na₂SO₃, anhydrous, 2.0 g.; hydroquinone, 0.3 g.; distilled water, 85 cc; acetone, 15 ml.). Wash thoroly in water and tone for 10 min. in 0.2% aqueous gold chloride, acidified with acetic acid. Wash in distilled water and reduce for 1 to 3 min. in 2% aqueous oxalic acid. Quickly rinse in distilled water and treat the sections 3 to 5 min. with 5% aqueous Na₂S₂O₃+5H₂O. Wash in water and stain overnight in Einarson's gallocyanin. Wash thoroly in water and place in 5% aqueous phosphotungstic acid for 30 min. From phosphotungstic acid transfer directly to a dilution (stock solution, 20 ml.; distilled water, 30 ml.) of the following stock staining solution: anilin blue, 0.01 g.; fast green FCF, 0.5 g.; orange G, 2.0 g.; distilled water, 92.0 ml.; glacial acetic acid, 8 ml.) and stain for 1 hour. Differentiate with 70% and 95% alcohol; pass the sections thru butyl alcohol and cedar oil; mount.     

Nongraded Dehydration and Low-Pressure Infiltration for Rapid Celloidin Embedding of Brain Tissue

Various ways of shortening single steps in the celloidin process have been combined to form a routine method which may be completed, for tissues of average size, within a week following fixation. Fixed, washed tissue slices 5 mm thick are dehydrated in 1 or 2 changes of absolute ethanol and acetone, 1:1. This requires 24 hr in an incubator at 37 C, or 12-16 hr if a magnetic stirrer is used. After ether-alcohol for 4 hr. the tissues are transferred to 5% celloidin and infiltrated in a vacuum desiccator attached to a filter pump. When the volume of celloidin is reduced to half the original amount (about 2 hr), the tissues are removed from the infiltrating fluid and embedded in 10% celloidin. The blocks are hardened in chloroform and cleared by suspending them in 2 or 3 changes of terpineol agitated by a magnetic stirrer. Sections are cut in terpineol, using any type of microtome. After washing in 95% alcohol, they are mounted on albumenized slides for staining.     

The Rapid Preparation of Celloidin Serial Sections Following India Ink Injections

For the study of capillary penetration in the central nervous system of the chick embryo, following India ink injections, celloidin serial sections are superior to those prepared by the paraffin technic. The celloidin sections are arranged on a moist cigarette paper mat, which when filled is inverted and applied to a microscope slide so that the sections contact the glass surface. Subsequent to dehydration and clearing the sections are isolated on the slide by peeling off the cigarette paper. Forty-five minutes are required to prepare a slide of thirty sections from the time the block is trimmed until the cover slip is mounted with Clarite.     

A Teflon Disk for Processing Loose Serial Celloidin Sections

Celloidin sections are routinely used for Nissl, Golgi, or Golgi-Cox staining (e.g., Glaser and Van der Loos 1981) when sections thicker than 30 μm are required. In spite of the advantages of the celloidin method (see Voogd and Feirabend 1981, Buschke 1979), processing free-floating serial sections of celloidin embedded material, which may often be preferred, is not very convenient.     

A Remodeled Tissue Flattener for Use with the International Microtome Cryostat

The antiroll plate is cut from a standard microscope slide, a 2 cm length, to give a 2 × 2.5 cm piece. This is fitted into inside grooves of a movable metal frame which is held by a hinge joint parallel to the back of the microtome knife. A stationary frame, which supports the hinged member, has spring clips welded to its sides for attachment to the knife. Clearance between the antiroll plate and knife is obtained by applying Scotch tape to the edge of the plate that is adjacent to the knife edge. The hinge permits the plate to be swung back and thus clear the knife surface.     

A Thermoelectrically Cooled Microtome Table and Knife

Thermoelectric cooling units (Frigistor thermoelements) have been used to replace CO₂ gas and solid CO₂ for microtome stage and knife cooling. These units consist of assemblies of series-connected thermoelements, functioning by the Peltier effect. Cooling is controlled by the direct current supply to the units. Current supply is from a double power pack giving 15 amp at 4.8 v for the knife cooling units, and 15 amp at 1 v for the stage. By varying the current flow, the optimum cutting temperature can be obtained and held indefinitely. An 8-couple Frigistor unit replaces the CO₂ stage of a Lipshaw freezing microtome. The stage temperature may be lowered to -36 C in 40-60 sec and at the optimum cutting temperature, 5 μ serial sections of fixed frozen tissue are obtainable. Four 12-couple units are used to cool a 160 mm Jung plane wedge microtome knife fitted to a Reichert sledge microtome, with the stage cooled by one 8-couple unit. The knife temperature can be lowered to -20 C in 5 min; the stage in 1 min. The apparatus has been used to cut a variety of unfixed rat and mouse tissues. The optimum sectioning temperature for such unfixed liver, kidney, spleen, lymph glands, heart, testes, small intestine, pancreas, skin and lung was -20 to -22 C.     

A Rapid Method for Macerating Phloem

Sieve cells and sieve tube members can be macerated from the phloem of various organs of woody and herbaceous species by au-toclaving the tissue in a mild macerating medium. This treatment does not digest the primary walls or the callose deposits on the sieve areas and sieve plates of the sieve elements. These cells can then be recognized by the fluorescence of their callose after staining with aniline blue. Sometimes adjacent sieve elements fail to separate and one can observe details of their junctures.     

Vitreous Carbon: A New Material for Making Microtome Knives

The quality of sections obtained by microtomy depends to a large extent on the quality and characteristics of the microtome knife itself. Despite the need for improved microtomy techniques, there have been few significant developments since the introduction of glass and diamond knives in the 1950's. The manufacture of microtome knives from vitreous carbon provides new possibilities for developing both improved methods and improved equipment for specimen sectioning. Vitreous carbon has unique physical properties that lend themselves to the generation of precision cutting edges. Such an edge can be obtained either by breaking a piece of vitreous carbon or by using lapidary techniques. The resultant edge seems well adapted to both thick and thin sectioning. The introduction of vitreous carbon as a sectioning tool offers a significant alternative to metal, glass and diamond knives.