A modified mallory-cason staining procedure for large cryosections.

The classic Mallory-Cason staining procedure has been modified for application to sections "on tape" obtained from large deep frozen tissue specimens. These 20 microns cryosections are collected on tape from a large heavy duty cryomicrotome. The stained sections provide anatomical details that are not revealed by other techniques. The merit of this procedure is found in the support of modern medical modalities, both for research and educational purposes.     

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.     

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.     

A Modified Mallory-Cason Staining Procedure for Large Cryosections

The classic Mallory-Cason staining procedure has been modified for application to sections “on tape” obtained from large deep frozen tissue specimens. These 20 μm cryosections are collected on tape from a large heavy duty cryomicrotome. The stained sections provide anatomical details that are not revealed by other techniques. The merit of this procedure is found in the support of modern medical modalities, both for research and educational purposes.     

Plastic Embedding, Orientation in the Mold and Tape-Supported Sectioning of Sclerotized Insects and Other Hard Objects

Sections of large specimens such as whole honeybees or beetle adults embedded in plastic usually are difficult to cut with a constant thickness. The sections also compress and roll. Sections of even thickness have been obtained by using a mixture of methacrylates (ethyl, 1:butyl, 3) and by firmly supporting the block in the microtome with a special holder. Scotch tape #810 applied to the block before each section is cut eliminates section compression and rolling. The sections are attached to slides with 2% celloidin in an absolute alcohol-methyl benzoate mixture (5:5-7:3); and the tape is removed with heptane. Large sections can also be cut from blocks of styrene mixed with butyl methacrylate. The specimens are oriented in the monomer in gelatin capsules by directing them into the desired plane among the fibers of a wad of absorbent cotton previously placed in the bottom of the capsule. The cotton is sectioned with the specimen but its fibers do not interfere, and remain outside the tissue.     

Tape transfer sectioning of tissue microarrays introduces nonspecific immunohistochemical staining artifacts

Abstract

Tissue microarrays place tens to hundreds of formalin fixed, paraffin embedded tissue cores into a paraffin block in a systematic grid pattern that permits their simultaneous evaluation in a single section. The fragmented nature of the tissue cores often makes sectioning of tissue microarrays difficult so that the resulting disks of tissue lose their shape, fracture or fall out of the paraffin section altogether. We have evaluated an alternative sectioning protocol for stabilizing the tissue microarray surface by placing an adhesive tape “window” over the face of the paraffin block prior to sectioning. Once sectioned, the tape/sections are transferred directly onto coated microscope slides, thereby avoiding routine floating of sections on a water bath. After sectioning with either the tape transfer or standard protocols, slides were stained either using hematoxylin and eosin or immunohistochemistry using antibodies to S-100 protein and the tissue specific antigens, keratin (AE1/3) and the leukocyte common antigen CD45. We found that the tape method produced thicker sections that were darker and more densely packed with loss of tissue definition compared to sections prepared using water bath flotation. Quantitative image analysis of immunohistochemical staining demonstrated that the tape method produced a higher incidence of nonspecific staining, which raised the potential for false positive staining.     

Enzyme histochemical reactions in unfixed and undecalcified cryostat sections of mouse knee joints with special reference to arthritic lesions

Summary The use of unfixed and undecalcified cryostat sections of mouse knee joints is described for the study of enzyme histochemical reactions. Non-inflamed knee joints and knee joints of mice with antigen induced arthritis have been used. Joints were embedded in gelatin and subsequently cut at low speed with a motor-driven cryostat fitted with a tungsten carbide knife at an obtuse angle (10°). The sections were attached to transparent tape to keep the integrity of the tissue intact. The following histochemical reactions were carried out succesfully: the tetrazolium salt reaction for dehydrogenase and reductase activity, the post-azocoupling method for acid phosphatase and cathepsin B activity and the simultaneous azo-coupling method for esterase activity. In all cases the morphology and integrity of the sections were well kept and serial sections were obtained without any difficulty. Nonspecific staining of the tape did not occur. The localization of the final reaction product was meeting criteria for specific and precise histochemical methods with the exception of the metal salt method because of nonspecific staining of undecalcified bone. Cytophotometry of the final reaction product appeared to be reproducible and valid as demonstrated by reaction for glucose-6-phosphate dehydrogenase activity in synoviocytes from knee joints with induced arthritis. End point measurements as well as kinetic measurements of the formazan production were performed and linear relationships were found between the specific formazan formation and section thickness or incubation time, respectively. It is concluded that cryostat sections attached to transparent tape are an excellent tool for the study of the metabolism in tissues adjacent to bone matrix. Changes of enzyme activities in synoviocytes, chondrocytes and osteoclasts during induced arthritis are discussed.     

Enzyme histochemical reactions in unfixed and undecalcified cryostat sections of mouse knee joints with special reference to arthritic lesions

The use of unfixed and undecalcified cryostat sections of mouse knee joints is described for the study of enzyme histochemical reactions. Non-inflamed knee joints and knee joints of mice with antigen induced arthritis have been used. Joints were embedded in gelatin and subsequently cut at low speed with a motor-driven cryostat fitted with a tungsten carbide knife at an obtuse angle (10 degrees). The sections were attached to transparent tape to keep the integrity of the tissue intact. The following histochemical reactions were carried out successfully: the tetrazolium salt reaction for dehydrogenase and reductase activity, the post-azo-coupling method for acid phosphatase and cathepsin B activity and the simultaneous azo-coupling method for esterase activity. In all cases the morphology and integrity of the sections were well kept and serial sections were obtained without any difficulty. Nonspecific staining of the tape did not occur. The localization of the final reaction product was meeting criteria for specific and precise histochemical methods with the exception of the metal salt method because of nonspecific staining of undecalcified bone. Cytophotometry of the final reaction product appeared to be reproducible and valid as demonstrated by reaction for glucose-6-phosphate dehydrogenase activity in synoviocytes from knee joints with induced arthritis. End point measurements as well as kinetic measurements of the formazan production were performed and linear relationships were found between the specific formazan formation and section thickness or incubation time, respectively. It is concluded that cryostat sections attached to transparent tape are an excellent tool for the study of the metabolism in tissues adjacent to bone matrix.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Hydraulic Powered Microtome in a Commercial Freezer; An Improved Cryostat for Large Sections

Modifications have been made on the Ullberg technique of taking whole-body sagittal sections of frozen mice on Scotch tape. Three improvements are described which greatly increase the ease and reliability of taking the sections. The microtome is driven by a hydraulic system for a smooth, dependable stroke. The microtome stage has been redesigned to eliminate uneven sections. The cryostat is an ordinary, commercial freezer of the frost-free design which eliminates the need for defrosting and also maintains a lower humidity.     

Autoradiography of Mobile, C14-Labeled Herbicides in Sections of Leaf Tissue

Fresh leaf tissue containing a soluble, C¹⁴-labeled herbicide was mounted in cold 1% gelatin on a holder, quick frozen in a cryostat, and cross sectioned at 16 μ with single-edge, stainless steel razor blades. The sections were transferred (without thawing) to cold (—10 C) microscope slides which had been partly covered with double-coated Scotch tape #665. The tissue was freeze-dried in a vacuum desiccator at—20 C then secured to the tape with pressure. Autoradiography was accomplished in a darkroom by covering the slides with dry, nuclear track emulsion films. These films were made by dipping 2 inch diameter wire loops into liquid emulsion, letting the film dry, and applying it by blowing it as it was placed against the tissue. After a 19 day exposure in light-tight boxes at 25-27 C the preparations were processed in the usual manner. The method-was used successfully to trace the movement of soluble, C¹⁴-labeled herbicides in leaf tissue without the loss of labeling material or artifacts caused by its diffusion. High resolution autoradiograms with low backgrounds were obtained.     

Squashing Under Scotch Tape No. 665 for Autoradio-Graphic and Permanent Histologic Preparations

Removal of the cover slip from squash preparations, for coating with auto-radiographic emulsion, or other purposes, is made easy if squashing is performed with a piece of Scotch double-coated adhesive tape No. 665, used as a cover slip. The material to be squashed is placed on a slide lightly coated with an adhesive consisting of 1% gelatin with 0.1% chrome alum added. The piece of tape is applied with the surface originally on the outside of the roll next to the specimen. Specimens should be soaked before squashing in aqueous 45% acetic acid, with or without added dye, such as carmine or orcein. After squashing, the tape is easily removed without damage to the cells. This allows autoradiographic emulsion to be applied, or, unstained material can be stained after squashing by technics suitable for microtome sections.     

The Use of Hydrophobic Adhesive Tape to Produce Miniature Wells on Microscope Slides

Hydrophobic adhesive tape was used to produce miniature wells on microscope slides for staining several sections of tissue with minimal amounts of cytochemical reagents. The wells could be tailored to individual specifications and the method allowed coverslips to be mounted close to the sections using either aqueous or xylene based mounting media. This method was especially useful for multiple immunolabelling of serial semithin cryosections.     

Autoradiography of Mobile,C14-Labeled Herbicides in Sections of Leaf Tissue

Fresh leaf tissue containing a soluble, C¹⁴-labeled herbicide was mounted in cold 1% gelatin on a holder, quick frozen in a cryostat, and cross sectioned at 16 μ with single-edge, stainless steel razor blades. The sections were transferred (without thawing) to cold (—10 C) microscope slides which had been partly covered with double-coated Scotch tape #665. The tissue was freeze-dried in a vacuum desiccator at—20 C then secured to the tape with pressure. Autoradiography was accomplished in a darkroom by covering the slides with dry, nuclear track emulsion films. These films were made by dipping 2 inch diameter wire loops into liquid emulsion, letting the film dry, and applying it by blowing it as it was placed against the tissue. After a 19 day exposure in light-tight boxes at 25-27 C the preparations were processed in the usual manner. The method-was used successfully to trace the movement of soluble, C¹⁴-labeled herbicides in leaf tissue without the loss of labeling material or artifacts caused by its diffusion. High resolution autoradiograms with low backgrounds were obtained.     

Conductive carbon tape used for support and mounting of both whole animal and fragile heat-treated tissue sections for MALDI MS imaging and quantitation

Analysis of whole animal tissue sections by MALDI MS imaging (MSI) requires effective sample collection and transfer methods to allow the highest quality of in situ analysis of small or hard to dissect tissues. We report on the use of double-sided adhesive conductive carbon tape during whole adult rat tissue sectioning of carboxymethyl cellulose (CMC) embedded animals, with samples mounted onto large format conductive glass and conductive plastic MALDI targets, enabling MSI analysis to be performed on both TOF and FT-ICR MALDI mass spectrometers. We show that mounting does not unduly affect small molecule MSI detection by analyzing tiotropium abundance and distribution in rat lung tissues, with direct on-tissue quantitation achieved. Significantly, we use the adhesive tape to provide support to embedded delicate heat-stabilized tissues, enabling sectioning and mounting to be performed that maintained tissue integrity on samples that had previously been impossible to adequately prepare section for MSI analysis. The mapping of larger peptidomic molecules was not hindered by tape mounting samples and we demonstrate this by mapping the distribution of PEP-19 in both native and heat-stabilized rat brains. Furthermore, we show that without heat stabilization PEP-19 degradation fragments can detected and identified directly by MALDI MSI analysis.     

Improved Trough for Ultramicrotomy

Troughs made of metal or tape have been used to provide liquid reservoirs for ultrathin sectioning with glass knives (Hayat 1970). However, these types of trough have certain limitations. the metal trough is difficult to use and its paraffin seal is easily broken, causing leaks. Hayat(1970) has even suggested that fumes from heating this paraffin may be carcinogenic. the tape trough is easier to seal but deforms easily while sections are being collected and does not always provide an even surface for the observation of interference colors. This report describes a method of making inexpensive plastic troughs that can provide a reservoir comparable to those used with diamond knives.     

A new optoelectronic measuring device for assessment of leg circumference in comparison with manual measurement]

For compression treatment to be effective in patients with chronic venous insufficiency, it is vital that leg circumference be measured accurately. If compression stockings are custom fit and appropriate for the medical indications, patient compliance will be high. Exact measurements of circumference and length are prerequisites for a good fit. The aim of the present study was to compare an opto-electronic device for the contact-free measurement of calf circumference with the conventional manual method using a tape measure. We investigated the differences between the results obtained with the two methods, and also their reproducibility. Circumferences were measured at defined heights on an anatomically shaped non-yielding leg model and on the leg of a healthy volunteer by 10 different experimenters both with the tape measure and with the opto-electronic device. The calf circumferences measured manually with the tape measure varied significantly more than those measured opto-electronically, both in the leg model and in the leg of the volunteer. A systematic error in the opto-electronic method appears unlikely, since the manual measurements on the leg model were both larger and smaller than those obtained with the opto-electronic device. Reproducibility was exceptionally high with the opto-electronic device (standard deviation 0.11-0.42 cm). The opto-electronic method yields rapid accurate measurements of circumference with excellent intra- and inter-operator reproducibility.     

Scanning Electron Microscopy of Pine Seedling Wood Tissue Sections Inoculated with the Pinewood Nematode Bursaphelenchus xylophilus Previously Prepared for Light Microscopy

Scanning electron microscopy (SEM) was applied to paraffin-embedded wood sections to study the histopathology of pine seedlings inoculated with the pinewood nematode (PWN), Bursaphelenchus xylophilus. The sections, which had been previously prepared and observed by light microscopy (LM) on glass slides, were originally obtained from experiments in which pine seedlings had been inoculated with PWN. The cover glass was removed by soaking the glass slide in xylene for 3 to 5 days. The glass slides were cut into small pieces so that each piece contained one wood section. Each piece of the glass slide was attached with double adhesive tape to an aluminum stub. The specimens were sputter-coated with gold and examined with a scanning electron microscope (JEOL-JSM 5200). Compared to LM (as documented in previous reports) SEM provided greater depth of focus and resolution of the damaged wood tissues, nematodes and associated bacteria. SEM made it possible to observe the relationship between bacterial distribution and nematode distribution in wood tissues. SEM observations also suggested the possibility of documenting the death of ray cells and other parenchyma cells in relation to disease development. Finally, the current study of PWN in pine seedlings demonstrated that glass slides prepared for LM observations more than 25 years earlier could be successfully processed for examination by SEM.     

Autoradiography of Water-Diffusible Substances in Sections of Whole Baby Rats

Rats, 7 days postnatal which had been injected with a radioactive nuclide, were quick frozen and sectioned in the frozen state. An adhesive cellulose tape (Sellotape) was used to support the section during cutting, through freeze-drying, and attaching to slides. Dehydration of the frozen sections consisted of 1 hr in a chilled desiccator containing silica gel, then at reduced pressure of 2-3 mm Hg until quite dry. The exposed side of the section was sprayed with celloidin dissolved in amyl acetate and allowed to dry. This side of the section was attached to a slide, previously coated with 1% gelatin containing 0.1% chrome alum, by means of an adhesive consisting of 4% gelatin and 5% formalin in 60% glycerol. In applying this adhesive it is mandatory that a border of about 3 mm of bare glass be left outside the adhesive, to allow intimate contact between the sticky side of the tape and the glass. The adhesive was allowed to set for 20 min, the slide immersed in water lor 50 sec, and the cellulose layer of the tape peeled off. The rubber base from the tape was removed with chloroform, the slide dried, and the exposed surface of the section coated with celloidin in amyl acetate, by dipping. After this treatment, the slides could be coated by dipping in autoradiographic emulsion without affecting water-soluble radioactive substances in the tissue.     

Preparing micro sections of entire (dry) conifer increment cores for wood anatomical time-series analyses

The type of samples most commonly used in dendro sciences are increment cores of conifers. These cores allow for an easy determination and measurement of ring-width variations over long time periods. For wood anatomical analyses, the cores have to be split into pieces to enable the preparation of high quality micro sections for detailed measurements of cell properties. A major drawback of this procedure is the fact that it is labor intensive and time consuming. We present a new technique enabling the preparation of micro sections of entire increment cores up to a length of 40 cm. For that purpose we combined standard wood-anatomical techniques with the application of Mowiol glue and common Tesa tape. We tested the introduced method on increment cores of Larix decidua Mill. sampled years ago for ring-width analyses to reanalyze them on a microscopic level. The ability to cut these long sections will tremendously reduce the time needed to prepare micro sections. This is of special interest for wood anatomical image analyses of cores used before to create long ring-width chronologies for any kind of environmental reconstructions.