The chemical determination of section thickness

Summary In quantitative histochemistry it is frequently desirable to know the exact thickness of the sections. The nucleic acid content of the tissue has been used as the basis of a method for determining section thickness. Such determinations agree remarkably well with the setting on the cryostat microtome although considerable variations in thickness can occur in sections cut at different speeds.     

Effects of tissue processing techniques in acoustical (1.2 GHz) and light microscopy

Summary In this study the influence of various tissue processing and staining techniques on the acoustical properties of liver tissue was investigated. A qualitative study was performed using ultrasound attenuation as the imaged parameter of a combined optical/acoustical microscope with a 1.2 GHz transducer. Images were made of three sets of adjacent liver sections (6 m in thickness) which were prepared in ten different ways: fixed by alcohol or formalin; stained by hematoxylineosin (HE), toluidine blue (TB) or non-stained; sectioned by a cryostat or by a paraffin microtome. It was concluded that the images obtained from cryostat sections were of much higher quality than those from paraffin sections. Images obtained from sections that were sectioned while embedded in paraffin displayed no detail at all. No consistent effect was noticed with respect to staining by HE or TB. Alcohol fixed sections gave more detailed images than formalin fixed sections. Formalin fixation in combination with cryostat sectioning yielded many cytoplasmic vacuoles.     

Comparison of two histological techniques for age determination in small cetaceans

Age estimation in odontocetes is based on counts of growth layer groups (GLGs) deposited in recording structures such as teeth. Generally, tooth sections are obtained using a cryostat microtome. However, some researchers prefer obtaining thin sections using a traditional paraffin microtome. Little information is available on the application of this technique to dolphin teeth. Our main aim was to investigate if the paraffin technique can be a viable alternative. We considered whether estimated age would be affected by preparation technique, staining method, and section thickness, while controlling for effects of species, body length, and sex. We also analyzed whether the staining method would affect readability of GLGs and age reading variability. Teeth from 86 individuals (representing seven species) were used, but not all were prepared using both techniques because sufficient teeth were not available in all cases. Although the staining method had significant effects on the estimated age using both techniques, the variability of GLG counts was small and appeared to be similar for both techniques. Using Mayer's hematoxylin stained sections of 8 μm thickness, good agreement of ages was obtained from both techniques, with more preparations classified as "good quality" for the paraffin technique. Mayer's hematoxylin provided the best contrast of the GLGs when using the paraffin technique. We conclude that the paraffin technique is viable and represents a cost-effective alternative to a cryostat microtome when preparing cetacean teeth for age determination.     

Staining method for whole-body autoradiography.

Sagittal whole-body sections of frozen mice were cut on a hydraulicly driven microtome in a cryostat at--15 C by applying cotton or nylon-backed adhesive tape to the mouse before cutting. Section thickness was 20 mu. The sections, still adhering to the tape, were dried in the cryostat (-15C) under atmospheric pressure. After autoradiography, the sections were pressed to a glass slide spread with a mixture of albumin and glycerin. The slide was immersed in xylene at 30 C for 15 min. The tape was then removed from the slide, where the section remained to be stained with hematoxylin-eosin. The section thus obtained enabled the tissue histology to be related to the autoradiogram. This method may also be applied to histochemical studies of substances insoluble in xylene.     

Effects of tissue processing techniques in acoustical (1.2 GHz) and light microscopy.

In this study the influence of various tissue processing and staining techniques on the acoustical properties of liver tissue was investigated. A qualitative study was performed using ultrasound attenuation as the imaged parameter of a combined optical/acoustical microscope with a 1.2 GHz transducer. Images were made of three sets of adjacent liver sections (6 microns in thickness) which were prepared in ten different ways: fixed by alcohol or formalin; stained by hematoxylin-eosin (HE), toluidine blue (TB) or non-stained; sectioned by a cryostat or by a paraffin microtome. It was concluded that the images obtained from cryostat sections were of much higher quality than those from paraffin sections. Images obtained from sections that were sectioned while embedded in paraffin displayed no detail at all. No consistent effect was noticed with respect to staining by HE or TB. Alcohol fixed sections gave more detailed images than formalin fixed sections. Formalin fixation in combination with cryostat sectioning yielded many cytoplasmic vacuoles.     

Further studies on section thickness measurement

Synopsis A rapid, simple, accurate and reproducible technique for measuring section thickness using an instrument known as a surfometer is described. It has shown that microtome settings cannot be relied upon as a means of monitoring the thickness of sections of quenched or wax-embedded tissue. However, there appeared to be a linear relationship between microtome setting and the thickness of dewaxed sections. Microtome settings seem to provide an accurate indication of the thickness of araldite sections.Paper given at the Royal Microscopical Society's European Histochemistry Meeting at Nottingham in September 1975.     

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.     

Fixation and embedding variables in the immuno-electron microscopic study of rat heymann nephritis

Summary Fixation and embedding variables were compared in immuno-electron microscopic localization of rat IgG in an autologous immune complextype nephritis. Specimens from kidney cortex were fixed for 3, 6 or 9 h in the following fixatives made in 0.1 M phosphate buffer at pH 7.44% paraformaldehyde, 2.5% glutaraldehyde, periodate-lysine-paraformaldehyde or modified Karnovsky's fixative. Localization of IgG was performed on tissue sections cut with a tissue chopper, cryostat or sliding microtome, using agarose, Ames O.C.T. Compound or polyethylene glycol respectively as cutting matrixes. The sections were incubated in peroxidase-labelled antirat IgG antiserum (diluted 120 with phosphate-buffered saline) for 60 h. Peroxidase activity was then revealed and the sections embedded in Epon. Exact localization of IgG throughout the sections and good ultrastructure were achieved when paraformaldehyde and agarose were used. Periodate-lysine-paraformaldehyde proved almost as useful as paraformaldehyde in connection with agarose in respect of peroxidase reaction and ultrastructure. Fixatives containing glutaraldehyde gave a mostly weak and unevenly distributed peroxidase reaction product. In the cryostat sections breaking of the tissue structure could not be avoided. When polyethylene glycol was used as cutting matrix no peroxidase reaction was achieved.     

Thickness measurements of skeletal muscle sections using the light microscope

Synopsis A new device is described for improving the accuracy of measuring the thickness of cryostat sections by the focusing technique in the light microscope. The necessity of such measurements is demonstrated by the great variation (range 2.55 m–11.93 m) in the thickness of serial cross-sections of frozen muscle biopsies from 12 healthy men. The final dehydration of the sections was found to reduce the thickness of fresh sections by 47%. However, dehydration caused the cross-sectional area to be reduced by only 2.8%.     

Demonstration of Insulin in Mammalian Pancreas by the Fluorescent Antibody Method

A method for the staining of mammalian pancreatic islets with a specific fluorescent antibody to insulin using frozen-dried sections rather than those cut with a cryostat is described. Sections were cut on a Servall microtome at 1-2 μ or when embedded in paraffin they were cut at 4-5 μ on a conventional rotary microtome. The latter procedure greatly simplified the search for islets.     

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

Summary 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: (1) appropriate supporting medium; (2) surface coating before each sectioning process, the coating being either non-permanent, permanent, or semi-permanent; (3) suitable techniques for affixing the coated sections to the slides using either pressure-sensitive adhesive or solvent-based adhesive; and (4) 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°C or a rotary microtome at room temperature.     

Determination of correction factors of 3H-β-self-absorption for quantitative evaluation of grain number in autoradiographic studies

Summary Deparaffinized and Feulgen-stained sagittal sections of the mouse brain were studied interferometrically in order to measure optical path differences of euchromatin and heterochromatin of various cell types. Furthermore, the ratio eu-: heterochromatin of each cell type was determined. From these data mass densities of karyoplasm and, finally, correction factors of ³H--self-absorption were calculated for comparing grain numbers of different cell types in quantitative autoradiographic studies after application of tritium-labelled substances. Remarkable differences of correction factors up to a factor of 2.18 were found. Furthermore, the actual section thickness was determined interferometrically. A reduction to about 0.60 of the microtome setting was measured in two different areas of the brain. Using mass densities together with actual section thickness correction factors for a thickness of 1 m were calculated. This was done also for cell types outside the brain the data of which were taken from literature. Thus, differences in correction factors up to about a factor of 4 were found pointing out the importance of considering ³H--self-absorption in quantitative autoradiographic studies.Dedicated to the sixtieth birthday of Prof. Dr. Brigitte Maurer-Schultze, Würzburg.     

A cytophotometric and electron-microscopical study on catalase activity in serial cryostat sections of rat liver

Summary The validity of the histochemical procedure for demonstrating catalase activity in cryostat sections of rat liver at the light-and electron-microscopical level was studied cytophotometrically. Incubations in the presence of 5 mm diaminobenzidine, 44 mm hydrogen peroxide and 2% polyvinyl alcohol performed on fixed cryostat sections resulted in the highest amounts of final reaction product precipitated in a fine granular form which was specific for catalase activity. Serial sections processed for electron microscopy indicated that the osmiophilic final reaction product was exclusively localized in the matrix and core of peroxisomes. The relationship between incubation time and the amounts of final reaction product generated by catalase activity as measured at 460 nm in mid-zonal areas of liver lobules showed non-linearity for the test-minus-control reaction because first-order inactivation of the enzyme occurred during incubation. Linearity of the test-minus-control reaction and section thickness was observed up to 8 m. Catalase in rat liver showed a K_m value of 2.0 mm for its substrate hydrogen peroxide when the diaminobenzidine concentration was 5 mm. It is concluded that the procedure for demonstrating catalase activity in serial cryostat sections of rat liver at the light- and electron-microscopical level is specific and can be applied to quantitative purposes. This approach may be useful in pathology, when only small biopsies are available, when the tissue is heterogeneous, and when other histochemical markers also need to be studied in the same material.     

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.     

New device for preparing thin slices of adipose tissue for metabolic studies in vitro

A new microtome is described which allows the rapid preparation of equal slices of well-defined thickness of fresh human tissue, especially adipose tissue. Presetting the microtome for a section thickness of 500 micro m, we found a variation of about 5% with human adipose tissue. Slices of human adipose tissue sliced by the microtome showed a higher sensitivity to insulin and a better reproducibility of results than slices prepared freehand.     

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.     

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.