Polyester Resin Embedding for Sectioning of Hard and Soft Tissues

Soft and calcareous tissues embedded in polyester resin may be cut on a sledge microtome to produce thin sections of 3-4 β thickness. Fixed tissues, dehydrated in ethyl alcohol, cleared in methyl benzoate and chloroform, are taken into a wide-necked bottle containing equal parts of polyester resin and chloroform with 0.75% catalyst. The bottle kept in water bath at 37°C is connected to a vacuum pump. With the evaporation of the chloroform under reduced pressure (approximately 10 mm Hg) infiltration is complete. Tissues transferred into a blocking form containing pure polyester resin with 1.5% catalyst are polymerized at 37° C until blocks are firm (48 hr or more). Blocks are prepared with at least 5 mm margin of plastic surrounding the tissue. The edge of the block adjacent to the knife is then filed at an angle of 45° to the cutting movement. Sections are cut with a wide-backed biplanar knife having a cutting edge of 40-44° positioned at an angle of 30° to the plastic block. As the resin is permeable to most stains, staining is carried out through the plastic Sections carried through staining procedures in wire baskets are floated onto slides and mounted in polystyrene; the cover-glass is compressed with a spring-clamp. Microscopic examination shows no staining of plastic, minimal shrinkage and good cellular detail.     

Evaluation of LR white resin for histology of the undecalcified rat tibia

Histology of plastic embedded undecalcified bone represents a challenging problem to the histotechnologist. We outline here an exploration of LR White resin as a suitable medium for histologic study of undecalcified rat tibia. A procedure was developed for light microscopy of rat tibia embedded in LR White and sectioned by sawing-grinding technics. The specimens were fixed in 10% neutral buffered formalin or alcohol-acetic acid-formol, dehydrated in ethanol, defatted in chloroform followed by resin infiltration and heat-curing of embedded blocks. The procedure of dehydration, defatting, infiltration, and polymerization can be completed within 10 days. Cold curing with accelerator provided by the manufacturer did not yield superior results compared to blocks cured with heat. Thick sections were obtained using a diamond wire saw, attached to plexiform slides, then ground and polished. Surface staining with Von Kossa silver reagent or toluidine blue revealed satisfactory morphological preservation of the mineralized bone sections. Artifacts like small bubbles appeared occasionally and could not be avoided despite prolonged infiltration or cold curing of blocks. Our method is relatively simple for base-line histologic study of rat tibia. The method offers advantages such as easy adaptability, reliable stainability, contrast, and resolution of bone architecture and marrow cells. Two other embedding media, Micro-Bed resin and Unicryl, were also tested, but produced inferior results.     

Epon Resin Infiltration and Immunogold Labelling of Pituitary Secretory Granules after Cryofixation versus Chemical Fixation

The degree of infiltration of epoxy resin into pituitary secretory granules was evaluated using X-ray microanalysis of the concentrations of chlorine in the epoxy resins. The effectiveness of infiltration was tested after three different tissue preparation techniques: cryofixation + freeze-drying (CF-FD), glutaraldehyde fixation (GF) + chemical dehydration, and no fixation— no dehydration. Signs of marked incomplete infiltration were found in embedded unfixed tissue while the other two techniques showed 80% infiltration. Uneven penetration was seen after CF-FD and GF. The plastic surface demonstrated a mountain-like appearance over the secretory granules after immunocytochemistry of the glutaraldehyde fixed tissue, whereas the CF-FD tissue showed a less furrowed surface. This probably is due to contact with water, which swells those parts of the granules that are unprotected by the plastic embedding medium. Our findings may explain why it is possible to perform immunocytochemistry on Epon embedded tissue.     

Method for the Prfservation of Polystyrene Latex Beads in Tissue Sections

The degree of infiltration of epoxy resin into pituitary secretory granules was evaluated using X-ray microanalysis of the concentrations of chlorine in the epoxy resins. The effectiveness of infiltration was tested after three different tissue preparation techniques: cryofixation + freeze-drying (CF-FD), glutaraldehyde fixation (GF) + chemical dehydration, and no fixation— no dehydration. Signs of marked incomplete infiltration were found in embedded unfixed tissue while the other two techniques showed 80% infiltration. Uneven penetration was seen after CF-FD and GF. The plastic surface demonstrated a mountain-like appearance over the secretory granules after immunocytochemistry of the glutaraldehyde fixed tissue, whereas the CF-FD tissue showed a less furrowed surface. This probably is due to contact with water, which swells those parts of the granules that are unprotected by the plastic embedding medium. Our findings may explain why it is possible to perform immunocytochemistry on Epon embedded tissue.     

The Use of Papain in Clearing Plant Tissues for Whole Mounts

Materials killed and fixed in FAA (formalin-acetic acid-alcohol) and similar fixatives frequently are difficult to clear for whole mounts because the denatured proteins will not become soluble in NaOH and other clearing agents. If tissues are washed for 3 days in running water, then incubated at 40 C for 5-7 days in 2% papain buffered to pH 7.2 and activated with 15 ml of .02 M Na₂S, cell contents are partly digested. Normal clearing with 5-10% NaOH followed by chloral hydrate (sat. aq.) can then effect complete solublility of cell contents and their removal. Permanent slides can be made after staining (1% safranin O in 50% alcohol for 12 hr is successful), by dehydration through alcohols, clearing in xylene, and mounting in resin.     

Resin tissue microarrays: a universal format for immunohistochemistry.

Tissue microarray (TMA) technology allows the miniaturization and characterization of multiple tissue samples on a single slide and commonly uses formalin-fixed paraffin-embedded (FFPE) tissue or acetone-fixed frozen tissue. The former provides good morphology but can compromise antigenicity, whereas the latter provides compromised morphology with good antigenicity. Here, we report the development of TMAs in glycol methacrylate resin, which combine the advantages of both methods in one embedding format. Freshly collected tissue fixed in -20C acetone or 10% neutral buffered formaldehyde were cored and arrayed into an intermediary medium of 2% agarose before infiltration of the agarose array with glycol methacrylate resin. Acetone-fixed resin TMA demonstrated improved morphology over acetone-fixed frozen TMA, with no loss of antigenicity. Staining for extracellular, cell surface, and nuclear antigens could be realized with monoclonal and polyclonal antibodies as well as with monomeric single-chain Fv preparations. In addition, when compared with FFPE TMA, formalin-fixed tissue in a resin TMA gave enhanced morphology and subcellular detail. Therefore, resin provides a universal format for the construction of TMAs, providing improved tissue morphology while retaining antigenicity, allows thin-section preparation, and could be used to replace preparation of frozen and FFPE TMAs for freshly collected tissue.     

Improved diethylene glycol distearate embedding wax

Diethylene glycol distearate wax and cellulose caprate resin, 4:1 respectively by weight, were melted together at 75 C for five hours with occasional stirring. The resin tempered the extreme brittleness of the wax without softening it, and raised the melting point only one degree to 50 C. Fixed plant tissues were dehydrated in ethanol, cleared in xylene, and infiltrated with wax. Modified diethylene glycol distearate was easier to trim and shape, and formed flat sections more consistently than the pure wax. Sections were cut singly on Ralph knives with attached water pools on an ultramicrotome. Sectionability was excellent at 2-3 micrometers, variable at 1.0 micrometer, but impossible at 0.5 micrometer. Sections were transferred onto water drops on slides, dried, dewaxed, stained, and coverglasses applied as in the paraffin method. Histological feature of plant tissues were much sharper in modified diethylene glycol distearate sections than in paraffin sections, and were similar to plastic sections.     

A modified adenosine polyphosphatase histochemical method to demonstrate Langerhans cells in fragile epidermis

Adenosine polyphosphatase enzymes provide useful markers for epidermal Langerhans cells. Established adenosine polyphosphatase histochemical methods were refined and applied to demonstrate Langerhans cells in thin sheets of murine dorsal epidermis. The skin was supported during staining by attaching the keratinized surface to polyallyl diglycol carbonate "plastic" slides with cyanoacrylate adhesive and flattening it with pressure from a glass slide on the dermal surface. Optimal specific staining of dendritic Langerhans cells occurred after fixation of ethylenediaminetetraacetic acid-separated epidermal sheets in cacodylate buffered formaldehyde for 20 min and incubation, in the presence of magnesium and lead ions, with 9.36 X 10(-4) M adenosine diphosphate (ADP) for 45 min. Better definition of the cells was obtained with ADP as a substrate than with any concentration of adenosine triphosphate.     

Infiltrating and Embedding Tissues with Mixtures of Polyethylene Glycols and Polyvinylacetate Resins

The use of water-soluble polyethylene glycol polymers (Carbowax, Hydrowax) as embedding media can be extended and facilitated by incorporating a water insoluble polyvinylacetate resin, AYAF (Union Carbide Co.). A combination of 7.5% resin added by heating to a 3:1 mixture of polyethylene glycols 1540 and 4000 gives blocks which may be cut at 2-3 μ. Sections can be floated and properly expanded on an ordinary water bath in a manner which may be impossible with Carbowax alone because of section fragility. This may require judicious adjustment of surface tension by the prior addition of minute quantities of the wax. On water, polyethylene glycol dissolves out of tissues, which remain supported by the resin. After attachment to albumen-coated slides, residual resin may, at option, be removed by a 1-2 min immersion in methyl alcohol without visible impairment of fat content. Abopon is used for mounting. The method appears suitable for the study of intracellular lipids, particularly in tissues which cannot be conveniently handled after Carbowax alone.     

Application and Evaluation of the Lead-Adenosine Triphosphatase Method in Skeletal Tissue

Application and evaluation of the lead-ATPase histochemical method in skeletal tissue has demonstrated an intracellular localization of enzyme activity. The skeletal tissue was demineralized for 72 hr in cold 10% aqueous EDTA adjusted to pH 7.2. Frozen sections were cut and placed on cold albumenized slides, oriented, thawed, dried in a cool air stream, and fixed for 10 min in cold (-2 to -3 C) 10% formalin buffered with Na-acetate and adjusted to pH 7.2. The sections were washed, treated with 10% EDTA for 20 min at room temperature, rewashed, and incubated for an optimal period of 30 min at 37 C. in the lead-ATP medium of Wachstein and Meisel. Following incubation the sections were washed, treated for 1 min with 1% (NH₄)₂S, rewashed, immersed for 30 min in 10% buffered formalin, dehydrated, cleared, and mounted. Evaluation of the substrate specificity suggests that other phosphatases associated with skeletal tissue do not complicate the ATPase reaction.     

A method for immunohistochemical detection of osteogenic markers in undecalcified bone sections

To evaluate the osteogenic potential of novel implant materials, it is important to examine their effect on osteoblastic differentiation. Characterizing the tissue response at the bone-biomaterial interface in vivo at a molecular level would contribute significantly to enhancing our understanding of tissue integration of endosseous implant materials. We describe here a new technique that overcomes difficulties commonly associated with performing immunohistochemistry on undecalcified sawed sections of bone. Sheep mandible specimens were fixed in an ethanol based fixative to maintain adequate antigenicity of the tissue. As a result, it was possible to omit antigen retrieval at high temperature for recovery of antigenicity, and detachment of sections from the slides was avoided. Following dehydration and infiltration, the specimens were embedded in a resin composed of polymethylmethacrylate and polybutylmethacrylate. Polymerization was achieved by adding benzoylperoxide and N,N-dimethyl-toluidine. This resin was selected because it maintained the antigenicity of the tissue, provided adequate properties for cutting 50 µm thick sections, and it facilitated deacrylizing the sawed sections. Acid-resistant acrylic slides were glued to the blocks using an epoxy resin based two-component adhesive to avoid detachment of the slides during the deacrylation procedure. Samples were stained for alkaline phosphatase, type I collagen, osteonectin, osteopontin, osteocalcin and bone sialoprotein. The EnVision + ™ dextran polymer conjugate two-step visualization system was applied for immunohistochemical detection of these bone matrix proteins. This procedure yielded positive staining for the osteogenic markers in cells and matrix components. The protocol described here facilitates the use of immunohistochemistry on resin embedded sawed sections of bone and provides a convenient and reliable method that can be used routinely for immunohistochemical analysis of hard tissue specimens containing implant materials.     

Basic Dye Counterstaining of Sections Impregnated by the Golgi-Cox Method

Celloidin blocks of Golgi-Cox impregnated material are cut at 50 μ, the sections collected in 70% alcohol, transferred to a 3:1 mixture of absolute alcohol and chloroform for 2 min, and then stored in xylene or toluene for at least 3 min, or up to 2 wk until processed further. Mounting is done on glass slides which have been coated with fresh egg albumen diluted in 0.2% ammonia water (or a 0.5% solution of dry powdered egg albumen) and then dried at 60°C overnight. For attachment to these coated slides, sections are first soaked for 2-3 min in a freshly prepared mixture of methyl benzoate, 50 ml; benzyl alcohol, 200 ml; chloroform, 150 ml; and then transferred quickly to the slides by means of a brush. After 2-3 min the chloroform evaporates and the celloidin softens. The slides are then immersed in toluene which hardens the celloidin and anchors the sections to the slides. Alcohols of descending concentrations to 40% are followed by alkalinizations, first in: absolute alcohol, 40 ml; strong ammonia water 60 ml, for 2 min, then in: absolute alcohol, 70 ml; strong ammonia water, 30 ml, for 1 hr. Excess alkali is then removed by 70% and 40% alcohol, 2 min each, and a 10 min wash in running tap water. Bleaching in 1% Na₂S₂O₃, for 10 min and washing again in tap water for 10 min completes the process preliminary to staining. The preparations are then stained for 90 min in an aqueous solution of either 0.5% cresylecht violet, neutral red, or Darrow red, buffered at pH 3.6. Dehydration and differentiation in ascending grades of alcohol, clearing with toluene or xylene, and applying a cover glass with a mounting medium having a refractive index of about 1.61 completes the process.     

Advantages in the Use of Aldehyde Fuchsin with Van Gieson's Picro-Fuchsin Counterstaining for Differentiating Bone and Cartilage

Specimens of bone were fixed in 10% neutral phosphate-buffered formalin or in Bouin's fluid and decalcified in 10% formic acid buffered with 10% sodium citrate. Materials were embedded in paraffin and 4-5 μ sections attached to slides were oxidized with 0.5% KMnO₄, decolorized in 1% oxalic acid, stained with aldehyde fuchsin, and counter-stained with Van Gieson's picro-fuchsin. Sections were dehydrated, cleared and mounted in a synthetic resin. Microscopically, the differentiation between bone and cartilage was seen as red and purple respectively, with connective tissue red; muscle and erythrocytes, yellow; and elastic fibres purple. The areas occupied by bone, cartilage and erythrocytes could be compared, and also the depth to which cartilage extended into the ossified sites. The advantages of this staining combination are: good contrasts in colour, ease of applying the stain, and virtual self-differentiation of the staining solutions.     

Agar-Screen Specimen Carrier for Bulk Processing of Biopsy Materials for Electron Microscopy

A specimen carrier for processing large numbers of biopsy materials for epoxy embedding and electron microscopy is described. Commercially available 18-mesh stainless steel or 16-mesh aluminum wire screening is used. The screening is cut into 1 × 3-inch strips. One corner is snipped off for orientation purposes. Four drops of warm 4% agar is placed on a prewarmed standard microscopic glass slide. A thin agar support film is formed on the bottom side of the horizontally held wire screen by lightly running it against the agar. Tissue blocks trimmed to 1 mm³ are blotted on filter paper and placed in a prearranged order on the top surface of the support film. A thin top coating of agar is applied on the specimen by touching it with the tip of a pasteur pipette containing warm 4% agar. The agar-screen unit with the mounted specimens is stabilized in 4% buffered formalin and rinsed with Sorenson's phosphate buffer, pH 7.4, with 6.8% sucrose. It is then processed as a unit through routine osmium tetroxide postfixation, alcohol dehydration, and Epon 812 infiltration. The tissue blocks are plucked off the agar support film with fine-tipped tweezers and embedded in individual capsules. No difficulty in thin sectioning was encountered and examination of the sections under the electron microscope showed good infiltration by the epoxy resin.     

Staining Procedures for Ultrathin Sections of Tissues Embedded in Polyester Resin

Tissues were fixed for 30 min In cold (0-2° C) 1% OsO₄ (Palade) buffered at pH 7.7, to which 0.1% MgCl₂ was added. Dehydration was in a graded ethanol series (containing 0.5% MgCl₂) at 0-2° C, and terminated with 2 changes of absolute ethanol. Tissues were then transferred by a graded series to anhydrous acetone. Infiltration of the tissue with Vestopal-W (a polyester resin), is gradual with the aid of graded solutions of Vestopal-W in acetone. The infiltrated tissue is encapsulated and initial polymerization is done under ultraviolet light at room temperature for 8-16 hr. This is followed by final hardening at 60° C for 36-48 hr. Sections (0.2-1 μ) were cut, dried on slides, placed in acetone for 1 min and then treated by either of the following staining procedures: (1) Thionin-azure-fuchsin staining: Flood the preparation with 0.2% aqueous thionin and heat to 60-80° C for 3 min; if the preparation begins to dry, add stain. Rinse in distilled water. Flood the slide with 0.2% azure B in phosphate buffer at pH 9. Heat to 60-80° C for 3 min; do not permit the preparation to dry. Rinse in distilled water. Dip the slide in MacCallum's variant of Goodpasture's carbol-fuchsin stain for 1-2 sec. Rinse in distilled water. Check the preparation microscopically for intensity of the fuchsin stain. Repeat dips as may be needed to obtain the desired intensity. Rinse in distilled water. Dehydrate quickly in 95% and absolute alcohol; clear in 2 changes of xylene and cover in Permount or similar synthetic resin. (2) Thionin-azure counterstain for the periodic acid-Schiff reaction: Oxidize the tissue in 0.5% periodic acid for 15 min and transfer to Schiff's leucofuchsin solution for 30 min. Counterstain with 0.5% aqueous thionin for 3 min; wash in distilled water; stain in 0.2% azure B in phosphate buffer at pH 5.5; wash in distilled water; dehydrate; clear and cover as in the first method. For temporary preparations let dry after absolute alcohol and apply a drop of immersion oil directly on the section.     

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.     

A Method for Histological Examination of Undecalcified Teeth

A method is presented for histological examination of undecalcified ground sections of tooth roots affected with periodontal disease. The roots were placed in Karnovsky's fixative overnight, postfixed in 2% buffered osmic acid, and dehydrated in an ascending series of ethanol. The specimens were then infiltrated with propylene-oxide and Epon-Araldite resin, embedded in Epon-Araldite, and sections were prepared using a cutting and grinding system. The resulting ground sections were 8-12 μm thick. The sections were allowed to air dry at room temperature. When thoroughly dried, a coverglass was applied using resinous mounting medium DPX. The specimens were examined by phase-contrast microscopy. The method is useful for simultaneous examination of mineralized dental tissue and bacterial morphotypes covering the root surface of teeth involved with periodontal disease.     

Novel Fixation of Plant Tissue, Staining through Paraffin with Alcian Blue and Hematoxylin, and Improved Slide Preparation

Onion (Allium cepa) root tips were fixed in a proprietary solution without aldehyde, toxic metals or acetic acid. Fixed specimens were embedded in paraffin, sectioned on a rotary microtome and mounted on detergent-washed slides without adhesive. Slides with ribbon segments affixed were immersed in 0.2% aqueous alcian blue 8GX in screw-capped Coplin jars in a water bath at 50 C for 1 hr. Excess alcian blue was rinsed off under cold running tap water and the slides were immersed in quick-mixed hematoxylin at room temperature for 15 min. Stained slides were deparaffinized, rinsed with isopropanol, air dried, and coverslips were affixed with resin. Thus, the traditional paraffin microtechnique has been modified at all steps from fixation to finishing slides with coverslips.     

Microscopy with Plastic Substitutes for Cover Glasses

To determine whether plastic substitutes for cover glasses on microscope slides affect the performance of the microscope, their optical constants were determined. The plastic covers and glass cover glasses were mounted also on silvered slides to form Star Test Plates which were studied by competent observers. The thicker plastic cover glasses, now on the market, are satisfactory when mounted to give a plane surface. Optically inhomogeneous materials, of irregular thickness, those that curl or do not have plane surfaces, adversely affect the performance of the microscope and should not be used. Since the substitutes are softer than glass they must be protected from abrasion. It is recommended that thicknesses of 0.18 mm., and none outside of a range of 0.12 to 0.20 mm. be used. For critical observation with unimmersed objectives of high aperture, best results are obtained with the correction collar set at the position corresponding to the actual thickness of the cover slip, just as would be done with glass cover glasses.     

Rapid Preparation of Pollen and Spore Exines for Electron Microscopy

Modifications in preparation techniques of acetolyzed pollen and spore exines for electron microscopy have reduced preparation time from the conventional 16-71 hr to 4-5 hr or less. These modifications include: (1) reduction of agar concentration from 4% to 0.9%; (2) omission of graded alcohol dehydration, going directly to acetone immersion and resin infiltration; (3) reduction of three steps in resin infiltration to one; (4) polymerization of resin at 80-85 C for 4-5 hr or at 90-98 C for 45-90 min, as opposed to conventional polymerization at 60-80 C for 12-59 hr.