Superficial Warming of Epoxy Blocks for Cutting of 25-150 μm Sections to be Resectioned in the 40-90 nm Range

An improved method is described in which tissue areas can be initially identified in thick sections by light microscopy and isolated for subsequent ultrathin sections and observation by electron microscopy. This is achieved by embedding in hard Epon which can be sectioned at 25-150 μm on a sliding microtome after softening the blockface by applying a 60-70 C tacking iron to its surface immediately before each section is taken. The thick sections are then mounted on plastic slides to enable light microscopic selection of areas to be observed by electron microscopy. The selected areas are remounted on faced Epon blanks and resectioned at less than 50 nm. This technique makes it possible to obtain thick sections while maintaining an Epon hard enough for good serial ultrathin sections.     

Adaptation of a sliding microtome for sectioning of material embedded in epoxy resins]

The employment of a sliding microtome of sectioning plastic embedded material with glass knives is described. Using a new knife holder and a modificated device for fixing plastic blocks succeeded in cutting sections 1--10 micron thick of relatively large pieces of tissue.     

Interferometric analysis of intrasection and intersection thickness variability associated with cryostat microtomy

Summary Mach-Zehnder interferometric measurements were used to assess the extent of section thickness variability (inter- and intrasection) associated with cryostat microtomy of adrenal sections over a typical working range of 10–20 m. Sections were obtained using a Bright's Cambridge rocking type and a Damon rotary type cryostat microtome to allow comparative analyses. The effective thickness of tissue sections after being mounted onto slides by flash drying was reduced by 90% relative to microtome section thickness setting. A linear relationship between measured thickness and microtome setting was obtained with both instruments. Thickness variability between replicate sections over the range of microtome settings approximated 11% for the rocking microtome and 5% with the rotary microtome. Average intrasection variability was found to be 7% for rocking microtome sections and 4% for sections obtained with the rotary microtome. However, this variability is a negligible source of error in cytophotometric analyses, providing replicate sections are used and an adequate number of measurements are made on mask-delimited individual cells or tissue specimen areas.     

Contributions to semithin sectioning on a conventional rotary microtome.

Procedures for obtaining sections 1 micrometer 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 holder 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 1 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.     

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.     

Frozen Section Micro-Incineration

A method for micro-incineration of frozen sections is described. Material containing diffusible or soluble salts is cut on the freezing microtome and the sections are placed into xylol and mounted out of xylol onto Corex D slides previously filmed with glycerin-gelatin medium. Material containing non-diffusible or insoluble salts can be fixed in 10% formalin before sectioning. Sections of the fixed material are dehydrated thru 50, 70, and 95% ethyl alcohol and mounted out of absolute alcohol onto Corex D slides previously fumed with glycerin-gelatin medium. After mounting by either procedure the sections are incinerated in an electric furnace and the temperature of incineration is dependent on the type of tissues to be incinerated and the character of the salts present. The method is time saving and when no fixation is required the whole procedure can be carried out in one hour.     

Sectioning and Staining Whole Heads of Small Animals

Serial sections showing minimal disruption in delicate structures of the heads of adult small vertebrates were produced with regularity when the heads were: (1) fixed in Bouin's picro-formol; (2) decalcified electrolytically to a radiographically-determined end point; (3) dehydrated in alcohols to 95%; (4) cleared in iso-amyl acetate; (5) infiltrated with concentrations (to 40%) of low viscosity nitrocellulose in iso-amyl acetate; (6) embedded in the same material by hardening it with chloroform; and (7) soaked in a mixture of 95% ethyl alcohol and glycerol (3:1) prior to sectioning on a sliding microtome. Following affixation to slides, the nitrocellulose matrix was dissolved from the sections and they were hydrated. Thin sections stained well with a standard Mallory technique. Thick sections were treated with the same stain, but good results depended upon empirical determination of times to be used in the procedure, and the introduction of water and 95% alcohol extraction-differentiation steps for red-staining and blue-staining tissues, respectively.     

Aerosol ot Solution—An Effective Softener of Herbarium Specimens for Anatomical Study

Dried plant parts are cut into convenient sizes and soaked in a solution containing 2.5-3.3% Aerosol OT in distilled water for 5 or more hours until well penetrated by the Aerosol. After brief washing in distilled water the material can be embedded, can be sectioned freehand or, if the nature of the material permits, with a sliding microtome, without embedding. Although it is a good wetting agent, Aerosol is chemically neutral; therefore, microchemical tests can be performed successfully on material treated with it. Refractory plant tissues embedded in paraffin can be successfully softened if one face of the block is trimmed to expose the tissue, then soaked in an Aerosol solution before sectioning.     

Holding Plastic Embedded Specimens During Dissection: Two Improvements

Sectioning of tissue specimens of aligned cells (e.g., muscle, cochlea, retina) for micrographs, often requires the capsule containing the tissue to be positioned at a precise angle during sectioning. The correct angle can be set by trial and error, but the process can be shortened if the gross anatomy of the cell system is used as a guide to orient the embedded sections as closely as possible in the optimal plane. Thick sections are then cut in this plane with a razor blade, and these sections are re-embedded in preparation for thin sectioning. This technique eliminates the large angles of the capsule in the microtome which occur when the gross tissue is poorly aligned in the first embedding.     

Improved Paraffin Sectioning with Etched Microtome Knife Edges

Carbon steel microtome knives etched with 0.1 N HNO₃ for 2 min display a very sharp cutting edge. Over a period of 3 yr no damage to the steel has been detected. The effect on paraffin sectioning was observed by comparing acid-treated knives with nonetched but well-sharpened ones. Sections of whole eyes cut with an etched blade showed approximately 15% less compression of the parffin matrix than those sectioned with an untreated knife. Tissues selected from routine autopsy material presented approximately 9% reduction in compression. As a result, excellent ribbons of sections could be cut from 5-7 μ and floated on water at 42—46 C with a minimum of folds or distortion. Etching improved sectioning when knife edges having bevel angles in a range of 31-39° were used, and also when the bevel was decreased to 20°, but the 20° edge gave impractically short service.     

A method for the preparation of serial thick sections of plastic-embedded plant tissues.

A procedure is described in which thick sections (2-10 mu or more) of plastic-embedded plant tissues are mounted in serial order on slides for use in routine light microscopy. Sections are cut with a steel knife on a rotary microtome while the block and blade are bathed with 40% alcohol. The cut sections are placed, in order, in 50% alcohol in the small wells of modified plastic trays where they become flat, pliable and suitable for subsequent handling. Sections remain separate and in correct order in the trays while they are stained, washed, and prepared for final mounting on slides. Mounting involves a simple and rapid procedure of transferring the sections to a slide and heating first on a 70-75 C hot plate (to slowly evaporate the water around the section and to partially affix the section) and then on a C hot plate. This second heating ensures adhesion when xylene-base mounting media, which tend to loosen weakly adhered plastic from the slides, are used. The technique of staining the sections loose provides the following advantages: (1) the problems of section loss and entrapment of stain between section and slide during staining are eliminated, (2) relatively high staining temperature, alkalinity, and alcohol concentration of the stain solvent (all of which promote loosening of pre-affixed sections from slides during staining) is allowed, and (3) staining is more even and selective. The procedure has been found to be reliable and fast enough to be of value in a significant variety of routine light microscope studies.     

Paraffin Section Thickness—A Direct Method of Measurement

Paraffin section thickness may be directly measured by re-embedding the sections wider consideration, cutting them again at right angles to the original plane of sectioning, and taking direct measurements with a filar micrometer after staining and mounting. Conditions and materials with which relatively un-distorted 3 and 5 μ sections were secured include (a) a hand-honed knife with a 23° facet bevel, set at a clearance angle of 7°, and (b) a hard paraffin (56-58°) embedding medium, preferably with 5% beeswax and 5% bayberry wax added. By taking direct measurements, it was found that bull testis tissue cut at a microtome setting of 10μ averaged 10.82 μ in thickness. Settings of 5 μ and 3 m resulted in sections averaging 5.25 and 3.31 μ in thickness respectively. Stages in sporogenesis of Onoclea sensibilis, Lewitsky fixed, were examined after sectioning at settings of 10, 5, and 3 μ to determine necessity for thin sections. For this material, it was indicated that mitochondrial preparations as thick as 10 μ were worthless, regardless of good fixation and proper staining. Three-micron sections give the best results.     

A Method for the Preparation of Serial Thick Sections of Plastic-Embedded Plant Tissues

A procedure is described in which thick sections (2-10μ or more) of plastic-embedded plant tissues are mounted in serial order on slides for use in routine light microscopy. Sections are cut with a steel knife on a rotary microtome while the block and blade are bathed with 40% alcohol. The cut sections are placed, in order, in 50% alcohol in the small wells of modified plastic trays where they become flat, pliable and suitable for subsequent handling. Sections remain separate and in correct order in the trays while they are stained, washed, and prepared for final mounting on slides. Mounting involves a simple and rapid procedure of transferring the sections to a slide and heating first on a 70-75 C hot plate (to slowly evaporate the water around the section and to partially affix the section) and then on a 100 C hot plate. This second heating ensures adhesion when xylene-base mounting media, which tend to loosen weakly adhered plastic from the slides, are used. The technique of staining the sections loose provides the following advantages: (1) the problems of section loss and entrapment of stain between section and slide during staining are eliminated, (2) relatively high staining temperature, akalinity, and alcohol concentration of the stain solvent (all of which promote loosening of pm-affixed sections from slides during staining) is allowed, and (3) staining is more even and selective. The procedure has been found to be reliable and fast enough to be of value in a significant variety of routine light microscope studies.     

Neurites and growth cones in the chick embryo. Enhanced tissue preservation and visualization of HRP-labeled subpopulations in serial 25-microns plastic sections cut on a rotary microtome

Study of axonal guidance in developing vertebrates has been hindered by an inability to readily visualize individual growth cones, determine the neuronal population from which they originate, trace their trajectories, and discern their interactions with their embryonic environment. We report a method that combines plastic embedding and serial sectioning with horseradish peroxidase labeling of subpopulations of neurons in the chick embryo. This method labels individual neurites from the soma to the tip of the growth cones, allowing their trajectory to be inferred and their identity to be determined by the position of the somata. As sections are up to 25 micron thick, entire growth cones can often be visualized without laborious reconstruction. Tissue preservation is much better than with similar material embedded in paraffin. Sections are cut relatively quickly using a steel knife on a standard rotary microtome and are suitable for subsequent electron microscopy.     

A new integrated concept for the improved preparation of sections of fresh or frozen tissue for light microscope histochemistry

In order to obtain thin sections of plant tissues which combined good morphological preservation and the preservation of the substances and enzyme activities in the tissues, a concept of section preparation by external stabilization was developed. The main components are as follows: appropriate supporting medium; surface coating before each sectioning process, the coating being either non-permanent, permanent, or semi-permanent; suitable techniques for affixing the coated sections to the slides using either pressure-sensitive adhesive or solvent-based adhesive; and mounting media with defined refractive indices (preferably UV-curable, water-soluble monomers). By this approach, sections exhibiting excellent morphological and physiological preservation were obtained using either a cryostat at -30 degrees C or a rotary microtome at room temperature.     

A simple method for studying whole sections of rice grain

We developed a new and simple method to collect sections of a whole brown rice kernel for investigation of histological properties. A single kernel of rice was dehydrated through a graded ethanol series, transferred to xylene, and embedded in paraffin. During sectioning of the blocks using a rotary microtome, we used a special adhesive tape to collect and place the sections on slides so they remained flat. The use of the adhesive tape technique combined with autofluorescence characteristics allowed us to visualize cell walls throughout an entire longitudinal or transverse section of a whole rice kernel. We obtained scanning electron microscopy images of the sections to determine section quality.     

A simple method for studying whole sections of rice grain

We developed a new and simple method to collect sections of a whole brown rice kernel for investigation of histological properties. A single kernel of rice was dehydrated through a graded ethanol series, transferred to xylene, and embedded in paraffin. During sectioning of the blocks using a rotary microtome, we used a special adhesive tape to collect and place the sections on slides so they remained flat. The use of the adhesive tape technique combined with autofluorescence characteristics allowed us to visualize cell walls throughout an entire longitudinal or transverse section of a whole rice kernel. We obtained scanning electron microscopy images of the sections to determine section quality.     

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.