A Technique for Embedding Undecalcified Bone Samples for Detecting Alpha-Emitters Using Vacuum Impregnation with Spurr's Resin

A method has been developed by which large samples of mineralized bone, containing an alpha-emitter, can be embedded in Spurr's resin in a fraction of the time required by conventional methods. Bone samples were freeze-dried or fixed and dried prior to impregnation with Spurr's resin under vacuum. Sections were cut for the preparation of either alpha-track or fission-track autoradiographs using the solid state detector CR-39. This method is applicable to samples containing a mobile form of a radionuclide that may be translocated during the processes of fixation and dehydration of the specimen.     

Embedding softened herbarium material in Spurr's resin for histological studies.

Plant organs, including stems, rhizomes, leaves, roots, petals, sporangia and flower pedicels obtained from dried herbarium specimens of a variety of plant species have been softened with Aerosol OT and subsequently dehydrated in a graded series of acetones and embedded in Spurr's resin. Although the quality of preservation varied, sections of a variety of materials showed excellent cellular preservation. Sections stained through the resin with toluidine blue O and examined with either bright field microscopy or with crossed polarizers showed good cell detail. Histochemical tests for callose, polysaccharides, and cellulosic walls, using sections from which the resin had been removed by sodium methoxide and then viewed with an epifluorescence microscope, gave excellent results.     

An Epoxy Resin Embedding Technique for Large Objects

Undecalcified heads in sizes up to that of a mallard are embedded, sectioned (10μ), and stained according to a modification of Luft's procedure (1961). Polymerization time is prolonged by keeping the resin mixture at 4 C or by omission of the accelerator. Under these conditions embedding takes about a month. The transparency of the blocks facilitates exact orientation of the object with respect to the sectioning plane, and milled reference grooves perpendicular to the sectioning plane permit exact reconstruction. Staining and mounting procedures are described. The technique has proved useful for exact determination of the spiritual disposition of the components of an object at a level between the microscopic and the anatomical.     

A Procedure for Embedding Plant Material in Araldite for Electron Microscopy

Plant material is embedded in Araldite made up according toLuft's compositional formula but with casting resin M replacedby type N. After fixation specimens are dehydrated rapidly inacetone of increasing 10 per cent concentrations. At the absolute-acetonestage specimens are left for I h with three changes of absoluteacetone during that time. Infiltration is initiated by replacinghalf the last change of absolute acetone by an equal volumeof Araldite mixture. Specimens are left to be infiltrated forat least I h on a rotary agitator. Half of the first infiltrationmixture is then replaced by the same volume of fresh Aralditeand the specimens left to agitate overnight. Specimens are thenembedded.     

Rapid Removal of Acid from Glycol Methacrylate for Improved Histological Embedding

A method for the rapid and complete removal of methacrylic acid from commercial samples of glycol methacrylate is presented. It entails conversion of the acid to an insoluble N-acylurea by treatment with an equivalent amount of N, N'-dicyclohexylcarbodiimide. Sections of tissue embedded in polymer prepared from the purified monomer can be stained with basic dyes without simultaneously staining the polymer.     

Immunohistochemistry on Resin Sections: A Comparison of Resin Embedding Techniques for Small Mucosal Biopsies

We have modified resin embedding methods to provide optimal information from en-doscopic biopsies. Mucosal biopsies were fixed either in buffered formalin and processed for embedding in Araldite or in acetone containing protease inhibitors and embedded in glycol meth-acrylate (GMA). GMA embedding generated an im-munophenotypic profile similar to that obtained in frozen sections while yielding far superior morphology and greater numbers of sections from small biopsies. The phenotypic markers included those for T cells, macrophages, mast cells, eosin-ophils and neutrophils. We have also demonstrated collagens, cell adhesion molecules and integrin molecules. Sections of similar quality were obtained with Araldite but the repertoire of antibodies was restricted to those which can be applied to formalin fixed, paraffin embedded tissues. We suggest that for optimal results, small biopsies to be subjected to immunochemistry are fixed in acetone at -20 C with the inclusion of protease inhibitors and embedded in GUIA with careful temperature control.     

Carbowax for Embedding and Serial Sectioning of Botanical Material

Plant material infiltrated with gradually increasing concentrations of Carbowax 400, followed by Carbowax 1540 and finally a 19:1 embedding mixture of Carbowax 1540 and 4000 showed minimum shrinkage. Quantitative measurements of shrinkage in tissue of potato tubers gave the following amounts: fixation and washing, about 4%; transfer from water directly to 70% Carbowax 400, 5176; from water through a graded series (5, 10, 15, 20, 30, 40, 50 and 60% Carbowax) to 70%, only 2.5% shrinkage; with an additional 1.5% occurring in transition to the embedding mixture. Dry ribbons are placed on adhesive-coated (gelatin, 5 gm; water, 120 ml; glycerol, 40 ml; phenol, 2 gm) slides in a humidity chamber. In 10-15 min enough moisture is absorbed by the ribbon to float the sections out gently and bring them in contact with the adhesive. Slides are then dried 5-10 min at room temperature. To remove minor wrinkles, the sections are subsequently flooded with water, then redried 12-24 hr; after which, they are ready for staining.     

Sandwich Embedding of Monolayers of Cells in Epoxy Resin for Ultrathin Sectioning

In this procedure for embedding monolayers of cells, the usual glass slides are replaced by plates of resin 1-1.5 mm thick. Unlike the open-face embedding technique, the present procedure uses only a few drops of unpolymerized resin, which are applied to the fixed and dehydrated cells. During polymerization this small amount of liquid resin spreads across a relative large area, leaves the cells covered by a very thin layer, and permits phase contrast observations through it. Ultrathin sections of a particular cell encircled by a rotary scriber can be obtained by sectioning the resin slide, which has been trimmed and mounted directly in the specimen holder of the ultramicrotome.     

Better Epoxy Resin Embedding for Electron Microscopy at Low Relative Humidity

In the absence of other factors known to influence sectioning properties, high environmental relative humidity is shown to yield poorly embedded tissue. Humidity-related effects are avoided if the following embedding precedure is used: impregnate tissues using the following solutions 1) 70% alcohol—5 minutes, 2) 95% alcohol—4 × 15 minutes, 3) absolute alcohol—3 × 40 minutes, 4) acetone—2 × 15 minutes, 5) 1:1 mixture of acetone-epoxy resin (DDSA, 63.4 g; Araldite 502, 5.6 g; Epon 814,39.4 g; DMP-30, 2.6 g)— 1 hour, 6) acetone-epoxy resin 13—1 hour, 7) epoxy resin—1 hour: complete the preparation of blocks as follows 8) when tissues have been oriented in epoxy resin in flat embedding molds, place molds in one evacuated vacuum desiccator 10 cm above a 2 cm layer of Drierite for 24 hours at room temperature, 9) raise temperature to 60 C and maintain for 3 days to cure resin.     

Growing and Embedding Fern Gametophytes for Autoradiographic Studies

Fern gametophytes were grown in liquid medium on the surface of plastic tissue culture flasks (Falcon Plastics) where they remained attached through radioactive precursor incorporation, fixation in buffered 3% glutaraldehyde, postfixation in buffered 2% OsO₄, alcoholic dehydration, and infiltration with an epoxy resin. Detachment of these plants from the plastic surface occurred only at the final step of infiltration with pure, unpolymerized resin. After detachment, the prothalli were kept in the resin to complete infiltration and then embedded. Sections 1-2 μm thick were cut, floated on a drop of glass-distilled water on clean slides and dried at 70 C. Kodak NTB-2 liquid emulsion was applied to the mounted sections and the emulsion-coated slides stored and developed according to established methods. The resulting autoradiographs showed excellent visualization of reduced silver grains, low background levels, and good preservation of cell structure.     

Resin Embedding for Quantitative Immunoelectron Microscopy. A Comparative Computerized Image Analysis

Quantitative immunoelectron microscopy (QIEM) is dependent on the reliability of preparative techniques for both efficient immunolabeling and consistent quantitative results among series of immunostained sections. The present study compared Lowicryl K4M and Epon embedding after identical fixation and dehydration of rat somatotrophic secretory granules. Labeling intensity, diameter, roundness, uptake of uranyl acetate, and gray value were measured with computer assisted image analysis. Lowicryl-embedded granules showed the highest labeling densities after conventional fixation and Progressively Lowering Temperature (PLT) dehydration, but values were not consistent in a series of immunostained sections. A lower but more uniform level of immunostaining was seen in Eponembedded sections when tissue was cryofixed and physically dehydrated. Gray value measurements from micrographs from both embedding media confirmed the better contrast of Epon sections and the different reliefs of the granule surfaces. This study emphasizes the importance of complete comparisons of preparative techniques for QIEM for reliability and reproducibility.     

Epoxy Embedding, Sectioning and Staining of Plant Material for Light Microscopy

By using a formula which gives a relatively soft epoxy embedding medium, it is possible to cut sections of plant material with a sliding microtome equipped with a regular steel knife. Blocks having a cutting face of 10 × 10 mm, giving sections of 4-10 μm, can be used. Tissues are fixed in Karnovsky's fluid, postfixed in 1 or 2% OsO₄, embedded in Spurr's soft epoxy resin, Araldite, or Epon mixtures. 5% KMnO₄, followed by 5% oxalic acid, then neutralized in 1% LiCO₃, are used to mordant the sections. Some of the stains used are Mallory's phosphotungstic acid-hemotoxylin, acid fuchsin and toluidine blue, or toluidine blue. Mounting is done with whichever soft epoxy resin was used in casting the blocks.     

Plastic Syringe Parts as Combined Centrifuge Tubes and Embedding Moulds for Suspended Material

If the shoulders and nozzle of a used plastic syringe be removed and the rubber plunger from the end of the piston inverted and replaced, a centrifuge tube is produced which may be used for agar or gelatin embedding of multiple small objects. The solidified block is removed by pushing up the plunger from below.     

Epoxy Embedding of Thin-Layer Material in Commercially Available Vinyl Cups for Light and Electron Microscopy

Epoxy embedding of cell cultures, impression smears and settled cell suspensions is performed in small vinyl cups, in which the entire sequence of culture, fixation, dehydration and embedding is feasible. The cup is readily stripped from the polymerized block to allow selected areas to be marked, photomicrographed by light, and thin sections of the selected parts cut for electron microscopy. The vinyl cups were obtained from Fabri-Kal Corp., Kalamazo, Mich. 49001 in sheets of 66 cups, each cup measuring 15 mm in diameter and 10 mm deep.     

An Embedding Method for Histochemical Studies of Undecalcified Skeletal Growth Plate

We have used glycol methacrylate to study undecalcified skeletal growth plate and subchondral bone. Minor modifications of the original technique including dehydration in glycol methacrylate vacuum infiltration and polymerization in the cold make it quite suitable for embedding of such tissues. Moreover, specimens can be processed quickly and the morphologic and biochemical integrity of the tissue retained so that histochemical procedures can be readily applied. Collagen, glycosaminoglycan, glycogen, lipid, calcium and the activity of alkaline and acid phosphatase were localized. This technique appears to be very useful for studying skeletal tissues.     

In Situ Embedding in Epoxy Resin of Tissue Cultured in Leighton Tubes; Selection of Single Cells for Electron Microscopy

Processing is carried out in the original culture vessel as follows: fixation in a 1.0% solution of phosphate-buffered OsO4 at 5 C, 30 min; dehydration in ethanol, 30 min; propylene oxide, 30 min; infiltration with a 1:1 mixture of resin and propylene oxide, 30 min; and undiluted resin at 5 C, 12 hr. This resin is replaced with 8 ml of fresh, and the tube is placed horizontally in an oven. Araldite is polymerized at 55 C for 21 hr; Maraglas, 60 C for 24-48 hr. Immediately upon removal from the oven, the hot tube is plunged into an ice-water bath at 0-0.5 C. The tube usually cracks extensively, and after 8-10 min soaking, the glass fragments can be easily removed. If a tube fails to break, it can be cracked mechanically, then allowed to soak until separation occurs. Advantages of the method are that an entire culture can be embedded, the resin cast separates cleanly, and any part of the culture becomes available for light and electron microscopy.     

A Method for Improved Resolution for Fluorescence Microscopy Using Plastic-Embedded Material Subjected to Resin Extraction

A protocol is given that uses NaOH, benzene, acetone and methanol to extract epoxy resins from semithin sections. Such sections appear superior to paraffin or unsectioned materials for fluorescence microscopic observations. Use of ultrarapid films (e.g., Kodak T-Max P3200) at ISO 3200 minimizes fading without use of antifading agents and without introducing unacceptable photographic grain size.     

Removal of Histological Sections from Glass for Electron Microscopy: Use of Quetol 651 Resin and Heat

A method is described for using the epoxy resin Quetol 651 and heat for convenient and rapid separation of conventional histological sections from glass slides for subsequent ultrathin sectioning for retrospective electron microscopy. The same method is useful when Epon-Araldite is substituted for the Quetol 651 resin.