A simple procedure to obtain one-micrometer sections of routinely embedded paraffin material

By freezing blocks of paraffin-embedded tissues to a convenient temperature it is possible to obtain routinely 1 micron sections that can be further processed as normal thicker sections. Normal and disposable steel knives can be used and the staining time should be increased in most procedures. Gradual freezing of blocks to the temperature of dry ice is the simplest and safest way to obtain an adequate temperature. The best results were obtained using as fixative 4% paraformaldehyde in phosphate buffered saline solution.     

A cryoprotection method that facilitates cutting frozen sections of whole monkey brains for histological and histochemical processing without freezing artifact

Cutting frozen sections of large (greater than 60 cc) blocks of monkey brain using the conventional procedures of infiltration with 30% sucrose as a cryoprotectant before freezing with pulverized dry ice often produces unacceptable levels of freezing artifact (FA) caused by displacement of tissue by ice crystals. Experiments investigating FA utilized perfusion-fixed brains from 46 monkeys and spanned combinations of cryoprotectants (glycerol, sucrose), freezing methods (dry ice or -75 degrees C isopentane), and fixatives (10% formalin, Karnovsky's or Timm's). The effects were evaluated by rating of FA severity in frozen sections of whole monkey brains. Minor FA appears as enlarged capillaries, more serious FA as large vacuoles, and both first appear midway between the periphery and center of the block. Stronger fixatives increased the severity of freezing artifact. The best method for eliminating FA was graded infiltration with up to 20% glycerol and 2% DMSO (in buffer or fixative), followed by rapid freezing in -75 degrees C isopentane. Although using a glycerol-DMSO infiltration before conventional freezing with pulverized dry ice or using conventional sucrose infiltration before freezing in isopentane gave better results than sucrose infiltration and dry-ice freezing, only the combination of glycerol-DMSO infiltration and freezing in isopentane produced consistently excellent results and virtually eliminated freezing artifact. To determine the effect of freezing with dry ice or isopentane on the rate of cooling in large blocks of CNS tissue, thermocouples were embedded in an 80-cc block of albumin-gelatin and frozen with the two methods. The rate of cooling (-3.5 degrees C/min) was twice as fast using isopentane.     

Some Improvements in the Preparation of Fresh Frozen Sections

For the histochemical demonstration of sensitive enzymes it is necessary to use fresh unfixed tissue sections. With the following procedure one can constantly obtain such sections 10-20μ thick with relative ease. Schanze's sliding microtome is employed. The microtome knife is deeply cooled by placing blocks of dry ice on its surface, and is provided with a device for preventing the sections from rolling up. The microtome is operated in an ordinary refrigerator maintained at a temperature of 0-3°C. For this purpose, the door of the refrigerator is replaced by a wooden door provided with a glass window, gloved arm holes, and a small door.     

A new convenient technique for making cell blocks

Cell block sections serve as an important diagnostic annex for cytological smears, liquid-based SurePath cytology and the Liquid-based Thin-prep Cytology Test (TCT). A variety of methods for the preparation of cell blocks are described in the literature and the techniques in cell blocks are in continuous improvement. A new technique for making cell blocks was introduced in the present study. We first used pregelatinized starch as the frame for the cell block, which is a really simple and economic method, because it can be carried out at room temperature without additional special instruments. We have performed hematoxylin and eosin (HE) staining, immunohistochemistry analysis and fluorescence in situ hybridization (FISH) in the cell block sections in 122 cytological specimens. The results demonstrated in this article show that pregelatinized starch is a useful frame for cell blocks. The pregelatinized starch can effectively collect even a few cells with powerful adhesiveness. Therefore, this new technique for making cell blocks is especially useful for cytologic samples with low cellularity, such as cerebrospinal fluid specimens.     

Multiple freezing points as a test for viability of plant stems in the determination of frost hardiness

A technique is presented for a simple, rapid, and reliable means of determining the viability of plant tissue subjected to freezing temperatures. Freezing curves of excised stems of Cornus stolonifera Michx., and several other genera were studied. Tissue temperature was recorded during freezing of plant stem sections. The heat of crystallization deflected the resultant freezing curves at points where tissue froze. Living stem sections of all genera studied revealed 2 freezing points, while dead tissue exhibited only 1. The influence of variables such as moisture content, sample size, thermocouple placement, and cooling rate on freezing curves was analyzed. Stem samples wrapped in moisture-proof film with a thermocouple inserted into the pith were frozen to a predetermined test temperature, thawed, and subjected to a second freezing cycle. The presence or absence of 2 freezing points in the second freezing cycle was used as a criterion for establishing viability. The results were immediately available and identical to results from regrowth tests which took about 20 days.     

An Application of the Frozen Sectioning Technic for Cutting Serial Sections Thru the Brain

The availability of CO₂ ice makes it practical to cut large blocks of cerebral tissue by the freezing method. If the tissue is first treated with 20-30% ethyl alcohol for sufficient time to secure uniform penetration of the alcohol (about 24 hours), formation of hard ice crystals can be controlled and serial sections 25-100 μ thick can be cut with negligible loss. The alcohol can be added to the fixative used for perfusion, or it can be added at any time later in the firing process, or after fixation is completed. The sections are cemented to the slide and groups of slides are manipulated thru staining processes in glass trays. Ordinary cell and fiber stains give satisfactory results. The method is particularly useful for certain neurophysiological purposes such as defining the location of electrode tracks and lesions and certain types of retrogrades. The Prussian blue test for electrolytically deposited iron can be conveniently applied in conjunction with other stains, to determine the point at which a given action potential response was observed, if steel electrodes are used.     

Ultrarapid vacuum-microwave histoprocessing

Summary A novel histoprocessing method for paraffin sections is presented in which the combination of vacuum and microwave exposure is the key element. By exploiting the decrease in boiling temperature under vacuum, the liquid molecules in the tissues have been successfully extracted and exchanged at relatively low temperatures during each of the steps from dehydration, clearing, and impregnation. In this vacuum-microwave method, an extremely short time suffices for the preparation of optimal-quality paraffin blocks. No xylene (but isopropanol instead) was used as the intermediate solvent. Thirty biopsies (thickness 2–4 mm) can be processed in 40 min. In addition, this approach can be used to produce large sections of giant blocks (4 × 6 × 1 cm³) which can be easily cut on a routine microtome due to the optimal paraffin impregnation. These giant blocks do not shrink during this vacuum-microwave histoprocessing.     

THE USE OF FORMALIN FIXATION IN THE CYTOCHEMICAL DEMONSTRATION OF SUCCINIC AND DPN- AND TPN-DEPENDENT DEHYDROGENASES IN MITOCHONDRIA

Brief formalin fixation in the cold prior to histochemical assay of rat liver and pancreas for various dehydrogenases has been used successfully to circumvent the structural damage and enzymatic loss to which mitochondria of frozen sections would otherwise be subject. To obtain an optimal result a single set of conditions has been devised, including fixation prior to freezing of minute (finely diced) organ blocks in graded concentrations (0.7 to 2.0 per cent) of formaldehyde in chilled (1–4°C) Hanks'' balanced salt solution, freezing at not higher than -70°C, and use of nitro-BT or, preferably, tetranitro-BT. The present histochemical study of hepatic and acinar cells indicates that not only are succinic and D-β-hydroxybutyric dehydrogenases located exclusively in the mitochondria but so are lactic, malic, and the isocitric dehydrogenases.     

The Rapid Preparation of Frozen Tissue Sections

The essential feature of this procedure involves the rapid freezing of the tissue following excision and keeping it frozen until the desired chemical or fixative has been applied. For freezing, either carbon dioxide or liquid air is used, as desired. The microtome knife is thoroughly cooled by taping blocks of dry ice to its surface. The cut sections, still frozen, are manipulated by a camel's hair brush so that they lie flat upon the knife. They are then transferred to a slide by a special section lifter. This has the form of a double-bottomed scoop packed with dry ice. Thus the section remains frozen while it is transferred to a clean microscope slide held at an angle above a Coplin jar of the desired reagent. The sections must be immersed just prior to melting. They curl and do not adhere to the slide if still rigidly frozen, and are distorted if immersed after melting.

With this technic sections showing a minimum of cellular distortion may be obtained. Consequently, it facilitates the use of many cytological technics, chemical tests, and enzymatic studies, such as the Gomori technics, on a variety of tissues.     

Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution

Transmission electron microscopy has provided most of what is known about the ultrastructural organization of tissues, cells, and organelles. Due to tremendous advances in crystallography and magnetic resonance imaging, almost any protein can now be modeled at atomic resolution. To fully understand the workings of biological “nanomachines” it is necessary to obtain images of intact macromolecular assemblies in situ. Although the resolution power of electron microscopes is on the atomic scale, in biological samples artifacts introduced by aldehyde fixation, dehydration and staining, but also section thickness reduces it to some nanometers. Cryofixation by high pressure freezing circumvents many of the artifacts since it allows vitrifying biological samples of about 200 μm in thickness and immobilizes complex macromolecular assemblies in their native state in situ. To exploit the perfect structural preservation of frozen hydrated sections, sophisticated instruments are needed, e.g., high voltage electron microscopes equipped with precise goniometers that work at low temperature and digital cameras of high sensitivity and pixel number. With them, it is possible to generate high resolution tomograms, i.e., 3D views of subcellular structures. This review describes theory and applications of the high pressure cryofixation methodology and compares its results with those of conventional procedures. Moreover, recent findings will be discussed showing that molecular models of proteins can be fitted into depicted organellar ultrastructure of images of frozen hydrated sections. High pressure freezing of tissue is the base which may lead to precise models of macromolecular assemblies in situ, and thus to a better understanding of the function of complex cellular structures.     

Effect of microtubule stabilization on the freezing tolerance of mesophyll cells of spinach

Freezing, dehydration, and supercooling cause microtubules in mesophyll cells of spinach (Spinacia oleracea L. cv Bloomsdale) to depolymerize (ME Bartolo, JV Carter, Plant Physiol [1991] 97: 175-181). The objective of this study was to determine whether the LT₅₀ (lethal temperature: the freezing temperature at which 50% of the tissue is killed) of spinach leaf tissue can be changed by diminishing the extent of microtubule depolymerization in response to freezing. Also examined was how tolerance to the components of extracellular freezing, low temperature and dehydration, is affected by microtubule stabilization. Leaf sections of nonacclimated and cold-acclimated spinach were treated with 20 micromolar taxol, a microtubule-stabilizing compound, prior to freezing, supercooling, or dehydration. Taxol stabilized microtubules against depolymerization in cells subjected to these stresses. When pretreated with taxol both nonacclimated and cold-acclimated cells exhibited increased injury during freezing and dehydration. In contrast, supercooling did not injure cells with taxol-stabilized microtubules. Electrolyte leakage, visual appearance of the cells, or a microtubule repolymerization assay were used to assess injury. As leaves were cold-acclimated beyond the normal period of 2 weeks taxol had less of an effect on cell survival during freezing. In leaves acclimated for up to 2 weeks, stabilizing microtubules with taxol resulted in death at a higher freezing temperature. At certain stages of cold acclimation, it appears that if microtubule depolymerization does not occur during a freeze-thaw cycle the plant cell will be killed at a higher temperature than if microtubule depolymerization proceeds normally. An alternative explanation of these results is that taxol may generate abnormal microtubules, and connections between microtubules and the plasma membrane, such that normal cellular responses to freeze-induced dehydration and subsequent rehydration are blocked, with resultant enhanced freezing injury.     

Cryostat Techniques: Methods for Improving Conservation and Sectioning of Tissue

Tissues have been conserved for satisfactory enzymatic histochemical assay for up to 12 mo by low temperature storage (in a dry ice chest or at -40° C) and by measures designed to offset the deleterious effects of sublimation of H₂O by using the following modifications of standard procedures (which include steps to give a mechanical support to otherwise fragmenting sections): 1. tissue blocks are coated with a polystyrene solution between each storage period; 2. modified bolts are used as tissue holders and types of bolt holders have been designed to fit on standard microtomes which permit manipulation of each tissue block independently of its mates on the same bolt holder, or the simultaneous cutting of all blocks on any one holder with each advance of the microtome feed; 3. tissues are coated with 20% polystyrene in methylene chloride prior to cutting and rubber cement painted on the slide as an adhesive. Lillie's 20% polystyrene diethylbenzene is used as a mounting medium. Other details of practical importance include the technique of freezing, control of moisture within a cryostat and on the microtome, and tests on histochemical procedures.     

光度法检测核桃青皮中类胡萝卜素含量

以核桃青皮为原料,浸提法获得萜类,用光度计对常温保存和冷冻贮藏下的类胡萝卜素含量进行分析。结果显示,常温保存和冷冻贮藏下,核桃青皮中类胡萝卜素含量差异很大。其中,常温保存时核桃青皮中类胡萝卜素含量为0．8％;冷冻贮藏时核桃青皮中类胡萝卜素含量为1．4％。结果表明,冷冻状态更有利于核桃青皮中类胡萝卜素的保存。     

Metallothionein as a clonable high-density marker for cryo-electron microscopy

Cryo-electron microscopy is expanding its scope from macromolecules towards much larger and more complex cellular specimens such as organelles, cells and entire tissues. While isolated macromolecular specimens are typically composed of only very few different components that may be recognized by their shape, size or state of polymerization, cellular specimens combine large numbers of proteinaceous structures as well as nucleic acids and lipid arrays. Consequently, an unambiguous identification of these structures within the context of a whole cell may create a very difficult challenge. On plastic-embedded specimens, or Tokuyasu sections (Tokuyasu, 1980), epitopes that are exposed at the surface can be tagged by antibodies. However, vitrified sections have to be kept at strict cryo-conditions (below -140 °C) and therefore do not allow any post-sectioning treatment of the specimens other than data acquisition in the microscope. Hence, the labels have to be placed into the specimen before freezing. Here we report on the application of a small metal-clustering protein, metallothionein (MTH), as a clonable label capable of clustering metal atoms into a high-density particle with high spatial resolution. We tested MTH as a label for kinesin-decorated microtubules (MTs) as well as the building blocks of desmin intermediate filaments (IFs).     

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.     

Cytophotometric DNA measurements of chondrosarcoma: methodologic aspects of measurements in tissue sections from old paraffin-embedded specimens

The methodologic prerequisites of cytophotometric DNA measurements of normal and tumor cells in tissue sections obtained from paraffin blocks and preserved as archival material were investigated. The optimal time of hydrolysis in 5-N HCl at room temperature was one hour for the different cell types analyzed. Paraffin-embedded tissues, stored for two decades, were still suitable for quantitative cytophotometric DNA determinations of Feulgen-stained nuclei. Different cell types in the Feulgen-stained sections could be identified with accuracy. The 90th percentile of fibroblast (internal control cells) DNA values was used as an upper limit of the diploid DNA content. By determining the number of tumor cells with DNA values exceeding this limit, nondiploid (hyperploid) tumor cell populations could be discriminated from diploid tumor cell populations. Cell population analysis of ploidy level, performed in this way, was found to be accurate in tissue sections of 4 micrometer. The accuracy of this analysis was not improved by increasing the section thickness. Since tissue sections obtained from old paraffin blocks could be used for the determination of hyperploidy, the prognostic significance of this parameter in different tumors can be assessed retrospectively.     

Thin sectioning of slice preparations for immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue. Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle. Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm. We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections. The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed. We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice. This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice. A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT). This is then quickly frozen with crushed dry ice and the resulting wafer can be position easily for cryostat sectioning. Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.     

Direct sectioning of unembedded cartilage: a simple method for microscopical and histochemical studies on chondrocytes and extracellular matrix

On account of the rigidity and compact structure of the hyaline cartilage, unfixed or formaldehyde fixed samples of this tissue can be directly sectioned by using a conventional ultramicrotome and a glass knife. This simple method allows to obtain microscopical sections from unembedded cartilage blocks, which show a well preserved histological structure and are very suitable to carry out morphological and histochemical studies on chondrocytes and cartilaginous matrix.     

The future is cold: cryo-preparation methods for transmission electron microscopy of cells

Our knowledge of the organization of the cell is linked, to a great extent, to light and electron microscopy. Choosing either photons or electrons for imaging has many consequences on the image obtained, as well as on the experiment required in order to generate the image. One apparent effect on the experimental side is in the sample preparation, which can be quite elaborate for electron microscopy. In recent years, rapid freezing, cryo-preparation and cryo-electron microscopy have been more widely used because they introduce fewer artefacts during preparation when compared with chemical fixation and room temperature processing. In addition, cryo-electron microscopy allows the visualization of the hydrated specimens. In the present review, we give an introduction to the rapid freezing of biological samples and describe the preparation steps. We focus on bulk samples that are too big to be directly viewed under the electron microscope. Furthermore, we discuss the advantages and limitations of freeze substitution and cryo-electron microscopy of vitreous sections and compare their application to the study of bacteria and mammalian cells and to tomography.     

Practical aspects of planning, building, and interpreting tissue microarrays: The Cooperative Prostate Cancer Tissue Resource experience

This is a review of several new approaches developed at or adopted by the Cooperative Prostate Cancer Tissue Resource (CPCTR) to resolve issues involved in tissue microarray (TMA) construction and use. CPCTR developed the first needle biopsy TMA, allowing researchers to obtain 200 or more consecutive cancer sections from a single biopsy core. Using radiographs of original paraffin blocks to measure tissue thickness we developed a method to produce TMAs with a larger number of usable sections. The modular approach to plan TMA construction is also a novel concept wherein TMAs of different types, such as tumor grade TMAs, metastasis TMA and hormone refractory tumors TMA can be combined to form an ensemble of TMAs with expanded research utility, such as support for tumor progression studies. We also implemented an open access TMA Data Exchange Specification that allows TMA data to be organized in a self-describing XML document annotated with well-defined common data elements. It ensures inter-laboratory reproducibility because it offers information describing the preparation of TMA blocks and slides. There are many important aspects that may be missed by both beginners and experienced investigators in areas of TMA experimental design, human subjects protection, population sample size, selection of tumor areas to sample, strategies for saving tissues, choice of antibodies for immunohistochemistry, and TMA data management.