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.     

Modification of a scanning transmission electron microscope specimen holder for large section viewing.

A modification of a scanning transmission electron microscope specimen holder which permits full viewing of large plastic embedded tissue sections is discussed. The method for producing one-centimeter diameter "giant" grids is explained and the procedure for sample preparation is outlined. The modification aids the microscopist in his evaluation of tissue structural relationships by providing large areas of tissue for examination and reduces significantly the time required to prepare and examine standard 1-2 mm2 electron microscopy tissue sections. Light and electron microscopic evaluations can be made on the same tissue sections.     

Histological Preparation of the Undecalcified Rat Otic Bulla for Mesoscopic Observations

Otic bullas of the rat, obtained by excision and formalin fixed, are successfully embedded in methylmethacrylate by dehydration and subsequent infiltration with plastic under vacuum. Sections 10 μm thick are obtained by cutting the trimmed and sandpapered acrylic blocks on an LKB multirange microtome. The sections are collected on adhesive tape and stained with a Trichrome stain (modified Weigert-van Gieson). Finally, the sections attached to the tape are mounted on microscope slides with glycerin-gelatin and sealed in the same medium. Serial sections are used for three-dimensional graphic reconstruction.     

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.     

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.     

Continuous Data Block Placement in and Elevation from Tertiary Storage in Hierarchical Storage Servers

Given the cost of memories and the very large storage and bandwidth requirements of large-scale multimedia databases, hierarchical storage servers (which consist of disk-based secondary storage and tape-library-based tertiary storage) are becoming increasingly popular. Such server applications rely upon tape libraries to store all media, exploiting their excellent storage capacity and cost per MB characteristics. They also rely upon disk arrays, exploiting their high bandwidth, to satisfy a very large number of requests. Given typical access patterns and server configurations, the tape drives are fully utilized uploading data for requests that fall through to the tertiary level. Such upload operations consume significant secondary storage device and bus bandwidth. In addition, with present technology (and trends) the disk array can serve fewer requests to continuous objects than it can store, mainly due to IO and/or backplane bus bandwidth limitations. In this work we address comprehensively the performance of these hierarchical, continuous-media, storage servers by looking at all three main system resources: the tape drive bandwidth, the secondary-storage bandwidth, and the host's RAM. We provide techniques which, while fully utilizing the tape drive bandwidth (an expensive resource) they introduce bandwidth savings, which allow the secondary storage devices to serve more requests and do so without increasing demands for the host's RAM space. Specifically, we consider the issue of elevating continuous data from its permanent place in tertiary for display purposes. We develop algorithms for sharing the responsibility for the playback between the secondary and tertiary devices and for placing the blocks of continuous objects on tapes, and show how they achieve the above goals. We study these issues for different commercial tape library products with different bandwidth and tape capacity and in environments with and without the multiplexing of tape libraries.     

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.     

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.     

Method for Staining and Washing Thin Sections on Support Films

A procedure is described for staining large numbers of thin sections on support films for use with one-hole grids. The film is picked up, carried and protected using easily made plastic blocks. Loop-tipped forceps are then used to transfer tissue ribbons from the knife boat to the support film. A large number of tissue sections can then be stained and washed simultaneously in a modified Pyrex dish without damaging the film. After staining, the slot in the one-hole grid is centered over the tissue ribbon, and the grid is attached to the film. The method is suitable for serial reconstruction and the unobstructed viewing of large thin sections in the TEM.     

Specimen preparation for quantitative microradiography

Summary The great importance of the pretreatment of biological objects for quantitative microradiography is discussed. Freeze-drying was proved a suitable method in obtaining dehydrated sections without any measurable change in the dry substance. The different steps of the freeze-drying procedure are analyzed with special reference to their effects on mass determinations. The most important errors are the effects of shrinkage and incomplete drying, their magnitude being discussed. For microradiographic determination of dry mass per unit volume the thickness of the freeze-dried tissue sections must be measured. Different methods for thickness determination are discussed and for freeze-dried sections a procedure for focusing in a microscope proved appropriate, the error in a single determination being less than 1 m.This work was supported by grants from the Swedish Medical Research Council (12X 587), Stiftelsen Therese and Johan Anderssons Minne, and Karolinska Institutet (Reservationsanslaget).     

Double-label immunocytochemistry: combination of anterograde neuroanatomical tracing with Phaseolus vulgaris leucoagglutinin and enzyme immunocytochemistry of target neurons

By the neuroanatomical tracing technique based on uptake, transport, and immunocytochemical detection of injected Phaseolus vulgaris leucoagglutinin (PHA-L), fiber trajectories of labeled neurons can be followed with great accuracy to their termination areas. To further analyze the connectivity of these fibers, the target neurons must be chemically characterized. In vibratome and frozen sections of rat brain, we tried to visualize PHA-L-labeled fibers and, simultaneously, the target neuron-related antigen. As a model system we used the projection from the pre-frontal cortex to histaminergic neurons in the posterior hypothalamic region. We tested "sequential" and "pooled" immunocytochemical procedures. In the sequential procedure, the two antigens are detected by two successive and complete immunocytochemical staining procedures, with primary antibodies raised in different animal species and with different chromogens for the final visualization. In the pooled procedure, the sections are incubated with mixtures of primary and secondary antibodies, after which the procedure is similar to the sequential procedure. We obtained excellent results on vibratome sections with a sequential procedure using first conventional peroxidase immunocytochemistry (goat anti-PHA-L primary antibody) to visualize the transported PHA-L (brown reaction product), and subsequently alkaline phosphatase immunocytochemistry (rabbit anti-histidine decarboxylase primary antibody) to locate the histaminergic neurons (blue reaction product). The resulting preparations deteriorate, however, after 1-2 months of storage. Good results were also obtained with a double peroxidase procedure on frozen sections, using nickel-enhanced diaminobenzidine to visualize the PHA-L (dark blue reaction product), and diaminobenzidine (brown reaction product) to visualize the second antigen. The quality of these preparations is permanent.     

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.     

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.     

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 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.     

Studies in lipid histochemistry

Summary Conditions for a selective extraction of nonpolar lipids from tissue sections with acetone were investigated using methods of lipid chromatography, histochemistry and quantitative determination of lipid phosphorus.Extraction of nonpolar lipids is selective when water (present either in acetone or in tissue sections) is completely excluded from the extraction procedure and the extraction is carried out at 0–4° C for 20–30 minutes. Under these conditions a negligible amount of polar lipids is extracted which cannot be demonstrated histochemically. A very small amount of nonpolar lipids remaining in sections cannot be demonstrated histochemically either.A method for the preparation of anhydrous acetone was recommended and an extraction procedure devised. This is to be applied in cases where nonpolar and polar lipids are to be distinguished and as an integral part of all types of phospholipid stains.When water is present during the extraction procedure (either in acetone or in tissue sections) significant extraction of all polar lipids occurs which is proportional to the content of water, to the length of extraction and to the temperature. Under these conditions the extraction of nonpolar lipids is somewhat slower.The significance of selective extraction with anhydrous acetone in histochemical analysis of lipids is discussed particularly with reference to lipids in atherosclerotic plaques.     

Quantitation of radio-ligand binding data using a desk-top calculator in the program mode

Quantitation of specific estrogen-binding capacity was facilitated using an inexpensive procedure for the rapid processing of data from radioligand binding measurements from sucrose gradient analyses. Ligand-binding data from each fraction of a gradient were punched into a paper tape in ASCII code and, later, read by a tape reader into an Olivetti Programma 101. Using the total instructional capacity (120 steps) a program was written in symbolic language which describes: (a) the calculation of counting efficiency of the instrument for each fraction, (b) the conversion of counts/minute to disintegrations/minute, (c) the computation of the concentration of [³H]estradiol-17β in each fraction and (d) the summation of the quantity of [³H]estradiol-17β bound by specified regions of the gradient. The program can easily be adapted for use with data generated from separation procedures such as centrifugation, electrophoresis or chromatography in which large number of samples are analysed.     

Mounting and Thionin Counterstaining of Fink-Heimer-Stained Sections of Dog Brain

The efficient use of thionin as a counterstain for large sections stained by the Fink-Heimer procedure (1967) requires mounting on slides before applying the thionin. The following modifications of the standard methods are recommended.     

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.