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.     

A Ralph Knife Holder Assembly for Use with the Sorvall “Porter-Blum” MT-1 Ultramicrotome

The superiority of plastic embedding for the production of high quality sections for light microscopy is well known, but the use of conventional glass knives with a cutting edge of approximately 4 mm has severely restricted the size of specimens in the past. Ralph knives provide a much longer cutting edge and adapters are available for certain models of microtomes and ultramicrotomes. A modified knife holder for use with the Sorvall “Porter Blum” MT-2 microtome was described by Gorycki and Sohm (1979); however, this is not suitable for the MT-1 model. We have therefore designed and made an adapter which enables Ralph knives to be used with this instrument. The design allows approximately 18 mm of cutting edge to be used on each knife, allowing larger specimens to be sectioned than with a conventional glass knife and reducing the frequency with which the knife needs to be changed when working with smaller blocks.     

A solvent-free coating-procedure for the improved preparation of cryostat sections in light microscope histochemistry

Summary A new technique is presented for the external stabilization of cryostat sections by spraying the specimen surfaces with an aqueous solution of poly(vinyl alcohol) before each sectioning stroke. The spray freezes upon the surface and forms a tough coating which facilitates subsequent sectioning and handling especially of difficult material. The sections are affixed upon cold glass slides covered with an improved formulation of pressure-sensitive adhesive. During further processing of the affixed sections, the PVA-coating and any surrounding supporting medium dissolve without traces in the first aqueous incubation or staining solution.     

A Simple Device to Help Re-Embed Thick Plastic Sections

The removal of epoxy capsules cast on glass slides is facilitated by 1) partial polymerization and brief exposure to elevated temperature, and 2) use of a slide holder to support the hot slides and reduce the chance of breakage. With this procedure, plastic sections routinely dried onto glass slides are available for re-embedding and subsequent thin sectioning.     

A solvent-free coating-procedure for the improved preparation of cryostat sections in light microscope histochemistry.

A new technique is presented for the external stabilization of cryostat sections by spraying the specimen surfaces with an aqueous solution of poly(vinyl alcohol) before each sectioning stroke. The spray freezes upon the surface and forms a tough coating which facilitates subsequent sectioning and handling especially of difficult material. The sections are affixed upon cold glass slides covered with an improved formulation of pressure-sensitive adhesive. During further processing of the affixed sections, the PVA-coating and any surrounding supporting medium dissolve without traces in the first aqueous incubation or staining solution.     

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.     

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.     

Some New Methods for Affixing Sections to Glass Slides. I. Aqueous Adhesives

As a new aqueous adhesive to affix sections to glass slides, hydrolyzed vinyl-triethoxysilane-either pure, in combination with polyvinyl alcohol or with specially prepared aqueous polyacrylate solutions-was applied. The silane proved to be very effective in enhancing bonding to the glass surface. As a general aqueous adhesive, a solution of 2% polyvinyl alcohol (m.w. 108,000; 99.7% hydrolyzed) with 0.2% hydrolyzed vinyltriethoxysilane is recommended. This stock solution is diluted 1:10 to 1:50 and used directly to float sections onto slides on a warming plate.     

Some new methods for affixing sections to glass slides. I. Aqueous adhesives

As a new aqueous adhesive to affix sections to glass slides, hydrolyzed vinyltriethoxysilane--either pure, in combination with polyvinyl alcohol or with specially prepared aqueous polyacrylate solutions--was applied. The silane proved to be very effective in enhancing bonding to the glass surface. As a general aqueous adhesive, a solution of 2% polyvinyl alcohol (m.w. 108,000; 99.7% hydrolyzed) with 0.2% hydrolyzed vinyltriethoxysilane is recommended. This stock solution is diluted 1:10 to 1:50 and used directly to float sections onto slides on a warming plate.     

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.     

Wrinkle-Free Plastic Sections for Light Microscopy

Plastic sections 0.5 to 2 μm thick are routinely used for light microscopy. Although plastic sections have several advantages over paraffin or celloidin sections, a problem that is often encountered with plastic sections is wrinkling (Fig. 1). Wrinkling occurs during staining when sections dried on glass slides are covered with stain and heated to hasten the penetration of the stain. Mounted sections heated on glass slides, but not stained, ordinarily lack wrinkles, even when examined with phase contrast optics. Similarly, mounted sections covered with stain, but not heated, lack wrinkles; unfortunately, such sections fail to stain adequately. Unmounted sections floated on heated drops of stain also lack wrinkles (Millonig 1980). Thus, it is clear that wrinkling occurs only when mounted sections are covered with stain and heated.     

Lamination of Thermoplastic Sheets as a Means of Mounting Histological Material

Permanent preparations of squashes, whole mounts and stained sections can be made by lamination of thermoplastic sheets. Classical procedures of staining and dehydration for sectioned material were used although dehydration after staining was not required for root tip squashes. Arranging the specimen with the identification label between two pieces of clear Vinylite plastic, 15 mils thick, tightening the preparation in a Photo-Seal Kit electric press and laminating for 3 min gave a finished preparation without the use of glass slides and cover slips. For root tip squashes, the stained tip was placed with a drop of stain between two pieces of plastic, squashed and then laminated. This insured retention of all the tissue which is sometimes lost during mounting processes. Preparations of unstained whole mounts were similarly laminated, with an identification label added between the plastic sheets. Stained sections were placed between two sheets of plastic but the identification label was placed on top of the preparation and a third piece of plastic added. This prevented the label from absorbing excess stain and the increased thickness allowed the slide to be used in a mechanical stage. Well preserved slides 18 mo old indicate that the laminated plastic slide is quite durable. It saves time, reveals good cytological detail and avoids some of the laborious features of other methods. It is a technic that can be used in introductory microtechnic and in the preparation of slides for class use in histology.     

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.     

Autoradiography of Water-Diffusible Substances in Sections of Whole Baby Rats

Rats, 7 days postnatal which had been injected with a radioactive nuclide, were quick frozen and sectioned in the frozen state. An adhesive cellulose tape (Sellotape) was used to support the section during cutting, through freeze-drying, and attaching to slides. Dehydration of the frozen sections consisted of 1 hr in a chilled desiccator containing silica gel, then at reduced pressure of 2-3 mm Hg until quite dry. The exposed side of the section was sprayed with celloidin dissolved in amyl acetate and allowed to dry. This side of the section was attached to a slide, previously coated with 1% gelatin containing 0.1% chrome alum, by means of an adhesive consisting of 4% gelatin and 5% formalin in 60% glycerol. In applying this adhesive it is mandatory that a border of about 3 mm of bare glass be left outside the adhesive, to allow intimate contact between the sticky side of the tape and the glass. The adhesive was allowed to set for 20 min, the slide immersed in water lor 50 sec, and the cellulose layer of the tape peeled off. The rubber base from the tape was removed with chloroform, the slide dried, and the exposed surface of the section coated with celloidin in amyl acetate, by dipping. After this treatment, the slides could be coated by dipping in autoradiographic emulsion without affecting water-soluble radioactive substances in the tissue.     

Some new methods for affixing sections to glass slides. II. Organic-solvent based adhesives

Adhesion of various organic-solvent based adhesives to glass slides could be greatly improved by first priming the slide with a copolymer of allyl methacrylate and methacryloxypropyltrimethoxysilane. The use of different solvents and types of adhesives with these slides is discussed. Cellulose nitrate in different esters of acetic acid proved to be an effective adhesive for varied sections at room temperature and in the cryostat. Carbowax sections as a special case preferably were affixed with polyisobutylene in petroleum ether. Most of the attachments formed resisted even boiling water.     

Some New Methods for Affixing Sections to Glass Slides. Ii. Organic-Solvent Based Adhesives

Adhesion of various organic-solvent based adhesives to glass slides could be greatly improved by first priming the slide with a copolymer of allyl methacrylate and methacryloxypropyltrimethoxysilane. The use of different solvents and types of adhesives with these slides is discussed. Cellulose nitrate in different esters of acetic acid proved to be an effective adhesive for varied sections at room temperature and in the cryostat. Carbowax sections as a special case preferably were affixed with polyisobutylene in petroleum ether. Most of the attachments formed resisted even boiling water.     

A Simple Method for the Selection of Specific Small Areas of Central Nervous Tissue for Electron Microscopy

An improved modification of an area or cell-selection technique is described. The method involves cutting 2.5-5.0 μm thick plastic sections, mounting than on 0.2 mm acetate sheet, examining them by phase-contrast microscopy, remounting selected sections and cutting these into ultrathin sections. Simplicity and speed are achieved by using acetate sheet instead of the usual glass slides and cover-slips. The method is suitable for topographic localization in small areas of the tissue and especially for the selection of dispersed single cells which are to be examined by electron microscopy.     

An Acrylic Spray Used as a Substitute for Cover Glasses

It has been found that a plastic spray (“Krylon”, manufactured by Krylon, Inc., 2601 Broad Street, Philadelphia, Pa.) is suitable as a covering medium for stained, paraffin-embedded tissue sections. The material is supplied in an aerosol bomb type dispenser. The technic and advantages of using a plastic spray to replace both the usual mounting medium and cover glass are described below.     

A new method for transfer of polyethylene glycol-embedded tissue sections to silanated slides for immunocytochemistry

Polyethylene glycol (PEG) is an excellent embedding medium for immunohistochemical studies. It provides structural preservation superior to frozen sections and increased sensitivity of antigen detection compared with paraffin sections. One limitation of PEG embedment is that PEG sections are difficult to handle and adhere poorly to glass slides. Here we present a simple and effective method for embedding tissues in PEG and transferring the resultant sections onto silanated glass slides. In addition, a method for silver enhanced colloidal gold immunostaining was combined with common dye staining to demonstrate the excellent structure preservation and sensitive antigen detection. Bovine chorionic membrane was fixed with Bouin's fixative, embedded in polyethylene glycol (PEG) 1500, cut into 5-microns sections, flattened over agarose blocks (10 x 10 x 2 mm3), and blotted onto Digene silanated slides. Slides were then washed in PBS, which removed the PEG and agarose blocks. Tissue sections were immunocytochemically stained with dilute antiserum raised in a rabbit against purified bovine placental retinol binding protein (bpRBP). Sections were washed and incubated with 1-nm colloidal gold-labeled goat anti-rabbit IgG. The immunogold particles were enhanced by silver staining (IGSS). Specimens were observed and photographed with an Olympus epipolarization microscope. The new method offered excellent morphological preservation of cell structure and the epipolarization microscopy provided high sensitivity for detection of specific immunogold-silver particles.