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 relationship 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.     

Notes on Technic: “Clearing” Steel Knife Epon Sections in a Polystyrene Film Sandwich

Serial sectioning epoxy embedments by steel knife permits rapid light microscope survey of large tissue volumes, and preselection of areas of interest for electron microscopy. Acetate film (Hollander 1970) and Turtox plastic slides (West 1972) have been suggested as substrates upon which the sections may be “cleared” with an added layer of cured epoxy. In our experience, these substrates are excessively adherent to Epon, and “cleared” sections thinner than 40-50 μm cannot be released from them reliably. The following method is suitable for processing Epon sections 10 or more microns thick.     

Prevention of Chemography in Prestained Epoxy Autoradiographs by an Intervening Carbon Film

Evaporation of a carbon layer over toluidine blue stained epoxy sections prior to applying emulsion eliminates or significantly reduces chemography that would otherwise be present in autoradiographs. This simple procedure permits routine proceasing of large numbers of slides without the limitations of the impermeable membranes currently recammended for light microscopic autoradiography following prestaining. The method permits “before and after” microphotography and use of simple staining procedures for sections to be studied autoradiographically.     

Reliable Preparative Procedures for the Cytological Study of Yolk-Laden Insect Eggs and Embryos

Yolk-laden insect eggs and embryos are easy to fix and section by the following procedure: 1) Fix with non- or slightly coagulant solutions after opening the envelopes for large eggs, or after superficial fixation and removal of the vitelline membrane for small ones. 2) Carefully embed fixed and washed specimens in a thick agar gel. 3) Dehydrate trimmed agar blocks, first with 70% ethanol, then with 2-ethoxy-ethanol. 4) After optional immersion in butanol-1, embed in Steedman's “Ester wax 1960.” 5) Section, mount and stain like paraffin sections. Results are compared to the cryosertioning method recently described by Hartmann.     

Detection of Radio-Elements in Histological Slides by Coating with Stripping Emulsion—The “Strip-Coating” Technic

The “strip-coating” technic offers some improvement of the published methods for the autographic detection of Tadio-elements in sections by stripping films. Reproducible results are easily obtained with Ilford Half Tone Stripping Plate Emulsion. The method does not lend itself readily to the preparation of the large number of slides usually required for biological research.     

A Rapid lmmunostaining Method for Frozen Sections

A simple and rapid one-step method for demonstrating immunohistochemical markers (leukocyte common antigen, cytokeratin, etc.) is described, which can help define the nature of poorly differentiated neoplasms for diagnosis using frozen section. Microwave irradiation was used to speed immunohistochemical analysis using “Enhanced Polymer One-step Staining” (EPOS) reagents on cryostat sections from a variety of pathologic samples. Reproducible results were obtained using EPOS reagents for leukocyte common antigen and cytokeratin. The overall procedure takes less than 10 min and can be completed during surgery.     

Improvements In The Paraffin Method

Dilute hydrofluoric acid alone and in conjunction with glycerin and ethyl alcohol was employed successfully to soften various types of refractory plant materials embedded in paraffin. Serial sections were cut at 6-8 μ and no appreciable deleterious effects on cell walls, cell contents, or staining procedures occurred. “Tannins” and “phlobaphene compounds” can be removed from tissues softened by this method by treating the sections for 12-48 hours with an aqueous solution of chromic acid, potassium bichromate and glacial acetic acid prepared according to the formula given by Johansen (1940).     

The Application of the Avidin-Biotin Technique to Previously Stained Histological Sections

Using the sensitive avidin-biotin peroxidase technique, a wide variety of tissue antigens can be detected in standard histological sections of both normal and pathological tissues previously stained with hematoxylin and eosin. There appears to be no detectable reduction of sensitivity with this method of “restaining” compared to the standard immuno-peroxidase procedure applied to unstained tissue sections. This technique makes it possible for retrospective identification of tissue antigens when insufficient unstained material is available.     

Preparing Mixed Cellulose Ester or Polycarbonate Membrane Filter Replicas for Transmission Electron Microscopy

Simple methods for preparing large numbers of grids exhibiting excellent coverage of intact replicas on mixed cellulose ester or polycarbonate membrane filters are described. The techniques ensure that grids and carbon replicas receive identical treatment and are not rearranged or lost during processing. The techniques permit grids and filter sections to be handled en masse rather than individually. Also, replica section squares remain centered on the grids. A temporary grid storage method (“grid- pad”) is also described, which facilitates grid identification and handling.     

Hot Melt “Corner Point Method” for Attaching Large Plastic Sections to Glass Slides

We describe a fast method for firm attachment of large plastic sections to glass slides with EVA-copolymers, commonly known as hot melt sticks. Solid hot melt sticks dissolve slowly in xylene to form an adhesive gel within 6 hours. Small drops of hot melt gel are applied to the corners of the sections and surrounding slide surface at ambient or elevated temperatures. The gel sticks to both the plastic and the glass slides. The hot melt “corner point method” prevented detachment of sections in staining procedures. As an additional technique, we suggest the use of hot melt adhesive for attaching plastic specimen blocks to wooden blocks or metallic specimen holders.     

Producing Frozen Sections of Calcified Bone

We describe a procedure for the rapid production and maintenance of fresh frozen bone biopsies which can be used for a variety of immunohistochemical techniques. Within 5 min of excision. tissue is placed in cold 5% polyvinyl alcohol, surrounded with 3% carboxymethylcel-lulose in a hand made aluminum foil embedding mold and frozen by immersion in an absolute ethanol/dry ice slurry at -70 C. The tissue block is attached to the specimen stub with cryocom-pound and installed in a -32 C cryostat whose tungsten carbide D profile knife is maintained at -70 C. Automatic controls are set at a slow cutting speed and the “sectioning window” is adjusted to fit the biopsy size. Knife angle, thickness gauge and antiroll bar are changed to produce a complete section. The block face is smoothly “papered” with a polyvinylpyrrolidone (PVP) impregnated Ross lens paper strip. A single section is cut and positioned on a sequentially numbered, acid cleaned, double dipped chrome-alum gelatin coated slide: adhesion is aided by “press-blotting” with bibulous paper. Sections are stored at -20 C or in a desiccator at room temperature. A brief fixation followed by removal of the water soluble PVP and lens paper generates fresh frozen bone sections suitable for further analysis.     

The Ultramicrotome Superstage: A Versatile Aid for Section Manipulation

The design and properties of a rigid, box-like device to be placed on the knife stage of a Porter-Blum MT-2 ultramicrotome are described. This “superstage” acts as a hand and tool rest, and a wind screen for the knife boat. The superstage easily permits tight rafts of ultrathin sections to be centrally located on grids. Additionally, it aids in placing 1 μm thick sections at the same relative location on glass slides to facilitate their examination in the light microscope.     

Stains for Strands in Sieve Tubes

Two very simple procedures give a staining-fixation of the so-called “strands” as well as portions of the sieve plates of sieve tubes of various broad-leaved deciduous trees. One procedure consists of placing hand-made sections (radial or tangential) of inner bark for 5 min in a 0.2% solution of ponceau S in 3% trichloroacetic acid, then soaking 5 min in 5% acetic acid. A second procedure consists of placing sections in 0.001% nigrosin in 2% acetic acid for approximately 15 hr, then washing briefly in distilled water. In the former procedure, strands, sieve plates, and what appears to be plasmalemma, appear reddish or pink, while cell walls do not stain. In the latter, strands and sieve plates appear bluish but phloem cell walls also become bluish, although xylem cell walls usually remain unstained.     

A Three-Dimensional Histological Method for Direct Determination of the Number of Trabecular Termini in Cancellous Bone

Osteoporotic fractures occur frequently in aging populations. Established methods for analyzing microarchitecture indicate that cancellous bone loss in the elderly is associated with progressive reduction in the connectivity of the trabecular network. This disconnection may explain the increased skeletal fragility that is sometimes out of proportion to the amount of bone lost. Connectivity, however, is difficult to measure and usually requires indirect methods. We describe development of a simple, inexpensive and direct procedure for counting sites of trabecular disconnection. The method is based upon preparation of 300-500 fjim thick slices of methylmethacrylate embedded material rather than the more usual thin 8 μm. histological sections. The marrow tissue is retained within the thick slice; this is essential for conservation of any detached bone fragments. In such preparations differential superficial staining of the upper and lower surfaces with alizarin red and light green, respectively, allows the two-dimensional image to be viewed at the same time as its three-dimensional counterpart. In this way, “real” (i. e., unstained) trabecular termini can be distinguished from “apparent” (i. e., stained red or green) termini that are artifacts of the plane of section. Partly polarized light enhances the microscope image. The method does not destroy the material for subsequent bone histomorphom-etry and, therefore, may be a useful adjunct to iliac bone biopsy analysis in studies of metabolic bone disease.     

An Inexpensive Apparatus for Cutting Tissue Sections on the Sliding Microtome by the “Dry Ice” Method

The use of “dry ice” in the preparation of frozen sections in histological technic has been increasing in popularity in recent years. Current literature largely deals with apparatus adapted for cutting large blocks of tissue, such as transverse or longitudinal sections of whole brains or other large organs, and usually must be constructed as a unit or in part by some particular manufacturer. The apparatus used by the author resembles that used by Dr. J. W. Lindsay, M.D.¹, but is less expensive and more easily constructed. The necessary materials may be found around the laboratory or may be purchased locally for not more than the total cost of twenty cents.     

Methenamine-Silver Staining: a Simple and Sensitive Staining Method for Senile Plaques and Neurofibrillary Tangles

An improved methenamine-silver impregnation method is presented which exhibits sensitivity for amyloid substances comparable to that of anti-β protein immunostaining. In optimally treated sections, this technique stained both β-amyloid deposits and neurofibrillary tangles, which are known to have a β-pleated structure. This simple procedure allows a large number of sections to be stained for routine examination.     

Embedding Plant Tissue with Plastic Using High Pressure: A New Method for Light and Electron Microscopy

An embedding technique has been developed to overcome difficulties that confront light and electron microscopists working with so-called “hard-to-embed” plant tissue. The method was originally described for freeze-dried material. It uses a modified Quickfit Rotaflo Valve and low heat to generate high pressure to aid in the infiltration and embedding of tissue with propylene oxide and plastic. The technique is not too cumbersome and requires 6 days from the dehydration step to the end of the polymerization process. Thick sections (1-2 μm) obtained from material prepared by this method stain readily with toluidine blue, and thin sections for the electron microscope stain satisfactorily following standard treatment with uranyl acetate and lead citrate. The thin sections are stable under the beam of the electron microscope. Results indicate that the quality of tissue preservation with this high pressure embedding technique is as good as that observed using standard embedding methods for electron microscopy.     

A Simple Method to Determine Hematoxylin Ripening

A simple spectroscopic method is proposed to control the “ripening” of Delafleld's hematoxylin solution during natural “ripening” or fast oxidation by different agents. The hematoxylin solution has ripened sufficiently for use with tissue sections staining if the absorption is 1.2-1.3 at 560 nm. The hematoxylin solution must first be diluted 15-fold using a 5% ammonium alum solution and the absorption must be measured in 0.5 cm cuvettes.     

Solochrome Cyanine R As An Indicator Dye of Bone Morphology

Sections of undemineralized bone embedded in a polyester resin and cut at 6 μ are stained for 10 min, without removal of the embedding matrix, in an aqueous solution composed of Solochrome cyanine R, 1 gin; glacial acetic acid, 2 ml; and distilled water, 98 ml. A pH about 2 is obtained by the acetic acid. The sections are washed and differentiated in tap water at 30 C, dehydrated in ascending alcohols, cleared and mounted in synthetic resin. “Young osteoid” stains light orange and, in the rest of an osteoid seam, two types of lamellae can be distinguished: one blue layer of ground substance or collagen and one orange layer of fibrillar collagen. The “calcification front” is sharply demarcated by its dark blue color.