Improved Polyester Wax Embedding for Histology

Polyester waxes are fatty add esters of polyethylene glycol. Polyethylene glycol 400 distearate melts at 35°C, infiltrates tissues well, and sections readily at 2 μ to more than 30 μ. Sections 2 μ to 6 μ are more easily cut when a kitchen strainer full of solid CO₂ (dry ice) is mounted above the microtome to cool the block and the knife, and when the knife crosses the block very slowly. Ribbons are flattened in water at room temperature and are mounted conventionally. Polyester ribbons are somewhat stickier than paraffin ribbons. Polyethylene glycol 400 distearate is slightly hydrophilic; immediately after microtomy and before the ribbon is affixed to the microscope slide, sections in the wax ribbon may conveniently be stained with 0.05% toluidine blue in aqueous benzoate buffer, pH 4.4. Tissue structure is better preserved in polyester than in paraffin wax, probably because structural lipids are better retained and localized. However, this difference between waxes is slight if tissues are well fixed and dehydrated. Other advantages of polyester wax are that sections fragment less, hard tissues rarely split away from the wax ribbon, no static electricity is generated, and the microtome knife seems to remain sharp for a longer time.     

Localization of plant lipids for light microscopy using p-phenylenediamine in tissues of Arachis hypogaea L.

p-Phenylenediamine (pPD) can be used en bloc to preserve and differentiate cell lipids in aldehyde-fixed peanut plant tissues treated with osmium tetroxide during dehydration in 70% ethanol. Semithin plastic sections for light microscopy need no further staining and can be mounted in Histoclad after drying on a slide. Brown staining above background differentiates lipid-containing structures. Nonspecific staining can be distinguished in control preparations extracted en bloc with lipid solvents.     

A Simple Device for Staining Many Single-Hole Grids Without Individual Manipulation

In serial sectioning for electron microscopy one of the greatest problems encountered is that the Formvar support film may break when grids are being mounted in or removed from a holder used for staining, or during staining. The latter is particularly troublesome when grids are stained individually. We describe here a device that conveniently eliminates this problem.     

A Method for the Preparation of Serial Thick Sections of Plastic-Embedded Plant Tissues

A procedure is described in which thick sections (2-10μ or more) of plastic-embedded plant tissues are mounted in serial order on slides for use in routine light microscopy. Sections are cut with a steel knife on a rotary microtome while the block and blade are bathed with 40% alcohol. The cut sections are placed, in order, in 50% alcohol in the small wells of modified plastic trays where they become flat, pliable and suitable for subsequent handling. Sections remain separate and in correct order in the trays while they are stained, washed, and prepared for final mounting on slides. Mounting involves a simple and rapid procedure of transferring the sections to a slide and heating first on a 70-75 C hot plate (to slowly evaporate the water around the section and to partially affix the section) and then on a 100 C hot plate. This second heating ensures adhesion when xylene-base mounting media, which tend to loosen weakly adhered plastic from the slides, are used. The technique of staining the sections loose provides the following advantages: (1) the problems of section loss and entrapment of stain between section and slide during staining are eliminated, (2) relatively high staining temperature, akalinity, and alcohol concentration of the stain solvent (all of which promote loosening of pm-affixed sections from slides during staining) is allowed, and (3) staining is more even and selective. The procedure has been found to be reliable and fast enough to be of value in a significant variety of routine light microscope studies.     

A method for the preparation of serial thick sections of plastic-embedded plant tissues.

A procedure is described in which thick sections (2-10 mu or more) of plastic-embedded plant tissues are mounted in serial order on slides for use in routine light microscopy. Sections are cut with a steel knife on a rotary microtome while the block and blade are bathed with 40% alcohol. The cut sections are placed, in order, in 50% alcohol in the small wells of modified plastic trays where they become flat, pliable and suitable for subsequent handling. Sections remain separate and in correct order in the trays while they are stained, washed, and prepared for final mounting on slides. Mounting involves a simple and rapid procedure of transferring the sections to a slide and heating first on a 70-75 C hot plate (to slowly evaporate the water around the section and to partially affix the section) and then on a C hot plate. This second heating ensures adhesion when xylene-base mounting media, which tend to loosen weakly adhered plastic from the slides, are used. The technique of staining the sections loose provides the following advantages: (1) the problems of section loss and entrapment of stain between section and slide during staining are eliminated, (2) relatively high staining temperature, alkalinity, and alcohol concentration of the stain solvent (all of which promote loosening of pre-affixed sections from slides during staining) is allowed, and (3) staining is more even and selective. The procedure has been found to be reliable and fast enough to be of value in a significant variety of routine light microscope studies.     

Localization of Plant Lipids for Light Microscopy Using P-Phenylenediamine in Tissues of Arachis Hypogaea L.

p-Phenylenediamine (pPD) can be used en bloc to preserve and differentiate cell lipids in aldehyde-fixed peanut plant tissues treated with osmium tetroxide during dehydration in 70% ethanol. Semithin plastic sections for light microscopy need o further staining and can be mounted in Histoclad after drying on a slide. Brown staining above background differentiates lipid-containing structures. Nonspecific staining can be distinguished in control preparations extracted en bloc with lipid solvents.     

Techniques for studying adipocytes

Various fixatives as well as tissue and slide handling procedures have been evaluated in attempts to demonstrate adipocytes histochemically while maintaining cell and tissue integrity. The optimal procedure for analysis of immature adipose depots consists of the following steps: 1) fresh, unfixed tissues are rapidly in isopentane quenched in a liquid nitrogen bath; 2) cryostat sections are cut, removed from the knife with a room temperature slide, and then air dried for 5-10 minutes; 3) slides can be stained directly with picro-Ponceau or toluidine blue procedures or with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-CaCl2 (1.25%). For analysis of mature rat adipose depots steps 2 and 3 are modified as follows: 2) cryostat sections are removed from the knife with a cold slide (-20 C) and dried for 30 minutes at 4 C; 3) the mounted sections are stained with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-HgCl2 (2.5%). When procedures described above for immature adipose depots are combined with esterase staining, adipocyte cytoplasm is clearly demonstrated. These procedures allow the routine use of fresh frozen, unfixed cryostat sections in studies of adipose cellularity.     

A Method for mass Preparation of Multiple Histopathological Slides

A method for the rapid preparation and staining of multiple mammalian tissues is described. With this method sections from 8-10 different organs can be handled simultaneously, mounted on the same slide, and stained with equally good differentiation and color contrast. It gives good results with either Zenker's or formalin-alcohol fixation.     

The labeled antigen method of immunoenzymatic staining.

A two stage immunohistological technique (the "labeled antigen" procedure) has been assessed for the detection of a variety of human and animal cytoplasmic constituents in tissue sections. In this method specific antiserum is followed by antigen complexed to horseradish peroxidase or to alkaline phosphatase. The primary antibody acts bivalently, linking the labeled antigen to antigen in the tissue section. The major advantage of this technique is that nonantigen specific antibody in the primary antiserum cannot cause nonspecific staining since it has no affinity for the antigen:enzyme complex. Consequently the specificity of the reaction is assured, background staining is minimized and the total staining time (from wax section to mounted slide) can be reduced to as little as 30 min. Further advantages include the possibility of labeling Ig allotypes and the high efficiency of enzyme utilization. Covalent human IgG:horseradish peroxidase complexes can also be used in a triple sandwich in conjunction with human anti-viral or autoimmune antibodies.     

Palladium chloride as a stain for elastin at the ultrastructural level.

Palladium chloride in aqueous solution stains elastic fibers in thin sections of Epon-embedded tissues. When palladium chloride is used with a lead citrate counterstain, high contrast sections with gray to black elastic fibers are obtained. The stain was tested on newborn and adult mammalian tissues and on adult tissues from lower animals. Sections were mounted on stainless steel grids, stained with 1% palladium chloride solution for 5 to 15 min, rinsed thoroughly, and counterstained with lead citrate for 7 min. Palladium chloride staining solution is stable for several months at room temperature and if the stain is filtered immediately before use, contamination of sections is not a problem. Chemical studies indicate that palladium binds directly to purified bovine ligamentum nuchae elastin and that this binding is not affected by glutaraldehyde fixation or by sodium borohydride reduction of elastin. Osmium post-fixation of glutaraldehyde-fixed elastin did significantly lower the amount of palladium bound. Palladium was shown to be chemically bound to sites on the elastin and not weakly associated. The nature of these sites is discussed.     

The application of polyethylene glycol (PEG) to electron microscopy

The cytoplasm of cells from a variety of tissues has been viewed in sections (0.25-1 micrometers) devoid of any embedding resin. Glutaraldehyde- and osmium tetroxide-fixed tissues were infiltrated and embedded in a water-miscible wax, polyethylene glycol (PEG), and subsequently sectioned on dry glass or diamond knives. The PEG matrix was removed and the sections were placed on Formvarcarbon-polylysine- coated grids, dehydrated, dried by the critical-point method, and observed in either the high- or low-voltage electron microscope. Stereoscopic views of cells devoid of embedding resin present an image of cell utrastructure unobscured by electron-scattering resins similar to the image of whole, unembedded critical-point-dried or freeze-dried cultured cells observed by transmission electron microscopy. All organelles, including the cytoskeletal structures, are identified and appear not to have been damaged during processing, although membrane components appear somewhat less distinct. The absence of an embedding matrix eliminates the need for additional staining to increase contrast, unlike the situation with specimens embedded in standard electron-scattering resins. The PEG technique thus appears to be a valuable adjunct to conventional methods for ultrastructural analysis.     

Improved Staining Boxes for Fast, Uniform Staining of Ultrathin Sections on Grids

A standard LKB (LKB-Produkter Ab. S161, 45 Bromma 1, Sweden) grid storage box is converted into several grid staining boxes by sawing the body of the box into segments along rows of its grid storage cavities. the staining boxes can be cut out to any required size or shape. the polymethacrylate storage box cover is discarded. Covers for the staining boxes are cut from thin sheet vinyl, which is more chemically resistant thin polymethacrylate. Corresponding 2 mm diameter holes are drilled through the vinyl covers and the bottoms of the grid storage cavities of the staining boxes to convert the storage cavities into staining chambers. for staining, the covers are tied to the boxes with sewing thread and the assembled units are put into vials. the separate staining chambers prevent intermingling of and mechanical damage to grids during the staining procedure. Ultrathin sections are more cleanly and uniformly stained in bulk by the use of these staining boxes than they are when stained individually by a standard method.     

A Method for Applying Different Stains to Alternate Serial Sections On a Single Microscope Slide

Every other section on the slide is coated with an inert grease (Dow Corning 7 Compound). The first staining technique is applied, thus staining the sections not covered with grease. The grease is dissolved in xylene, the slide rehydrated, and the sections already stained are coated with grease. The second staining technique is applied, and the grease is again dissolved in xylene, yielding finally a slide on which alternate sections have been stained by the two different staining techniques. Silicone greases, applied with a syringe and blunt hypodermic needle, are useful coating materials because of their insolubility in water and alcohol, chemical inertness, and heat stability. Silicone coatings are compatible with most staining techniques, including those which use strong oxidants, reductants, acids, bases, enzymes, and high temperatures. The method is particularly useful in examining serial sections of small blocks of tissue, or of tissues varying significantly in structure every few sections.     

Improved staining boxes for fast, uniform staining of ultrathin sections on grids.

A standard LKB (LKB-Produkter Ab. S-161, 25 Brommma 1, Sweden) grid storage box is converted into several grid staining boxes by sawing the body of the box into segments along rows of its grid storage cavities. The staining boxes can be cut out to any required size or shape. The polymethyacrylate storage box cover is discarded. Covers for the staining boxes are cut from thin sheet vinyl, which is more chemically resistant than polymethyacrylate. Corresponding 2 mm diameter holes are drilled through the vinyl covers and the bottoms of the grid storage cavities of the staining boxes to convert the storage cavities into staining chambers. For staining, the covers are tied to the boxes with sewing thread and the assembled units are put into vials. The separate staining chambers prevent intermingling of and mechanical damage to grids during the staining procedure. Ultrathin sections are more cleanly and uniformly stained in bulk by the use of these staining boxes than they are when stained individually by a standard method.     

Histological Technic for Cerebellar Climbing and Mossy Fibers

In order to, avoid disadvantages attendant upon the use of fresh frozen sections, or of block impregnation with silver, in staining climbing or mossy fibers of the cerebellum, Rio Hortega's double impregnation method for nerve fibers is useful. This consists of prolonged formalin fixation prior to cutting frozen sections (which thereafter are easier to cut) and preliminary treatment with ammoniacal aqueous and alcoholic washes, mordanting in pyridine silver, and treatment with pyridine-silver-carbonate. Following this, sections are handled individually through one of several reduction methods after which they may be directly mounted or gold toned.     

Isolation of Avian Trypanosomes by Selective Centrifugation and Subsequent Processing for Electron Microscopy

Paraffin sections are usually rehydrated before staining. It is possible to apply aqueous dye solutions without first removing the wax. Staining then occurs more slowly, and only if the embedding medium has not melted or become unduly soft after catting. To avoid this problem, sections are flattened on water no hotter than 45 C and dried overnight at 40 C. Minor technical modifications to the staining procedures are needed. Mercury deposits are removed by iodine, and a 3% solution of sodium thiosnlfate in 60% ethanol is used to remove the iodine from paraffin sections. At room temperature, progressive staining takes 10–20 tunes longer for sections in paraffin than for hydrated sections; at 45 C, this can be shortened to about three times the regular staining time. After staining, the slides are rinsed in water, air dried, dewaxed with xylene, and coverslipped in the usual way. Nuclear staining in the presence of wax was achieved with toluidine blue, O, alum-hematoxylin and Weigert's iron-hematoxylin. Eosin and van Gieson's picric acid-acid fuchsine were effective anionic counterstains. A one-step trichrome mixture containing 3 anionic dyes and phosphomolybdic acid was unsuitable for sections in wax because it Imparted colors that were nninformative and quite different from those obtained with hydrated sections. Advantages of staining in the presence of wax include economy of solvents, reduced risk of overstaining and strong adhesion of sections to slides.     

Contributions to semithin sectioning on a conventional rotary microtome.

Procedures for obtaining sections 1 micrometer thick on a conventional rotary microtome are described. Hydrophilic resin blocks with adequate hardness and elasticity for semithin sectioning are made by addition of divinylbenzene and methylmethacrylate to a commercial embedding kit. The blocks are pinched between two simple adapters and mounted in the specimen holder of a microtome. A glass knife of the Ralph type with an effective blade length of 25 mm is made from a glass slide and attached to a metal bar with paraffin. The low cost assembly is set in the steel knife holder of a conventional rotary microtome. Sections 1 micron in thickness can be cut from the resin embedded blocks. Staining with the usual staining solutions may be weak due to the thinness of the sections, but the fine resolution and low distortion achieved are compensating gains.     

Contributions to Semithin Sextioning on A Conventional Rotary Microtome

Procedures for obtaining sections 1 μ thick on a conventional rotary microtome are described. Hydrophilic resin blocks with adequate hardness and elasticity for semithin sectioning are made by addition of divinylbenzene and methylmethacrylate to a commercial embedding kit. The blocks are pinched between two simple adapters and mounted in the specimen bolder of a microtome. A glass knife of the Ralph type with an effective blade length of 25 mm is made from a glass slide and attached to a metal bar with paraffin. The low cost assembly is set in the steel knife holder of a conventional rotary microtome. Sections I micron in thickness can be cut from the resin embedded blocks. Staining with the usual staining solutions may be weak due to the thinness of the sections, but the fine resolution and low distortion achieved are compensating gains.     

A Scanning Electron Microscopic Evaluation of Section and Film Mounting for Transmission Electron Microscopy

Mounting of support films and sections for transmission electron microscopy has been examined with the scanning electron microscope. Experiments have been designed to test the adherence of support films to polished and matte surfaces of specimen grids. It is the conclusion of the authors that sections and films should be mounted on the dull or matte surface of Athene-type specimen grids.     

Techniques for Studying Adipocytes

Various fixatives as well as tissue and slide handling procedures have been evaluated in attempts to demonstrate adipocytes histochemically while maintaining cell and tissue integrity. The optimal procedure for analysis of immature adipose depots consists of the following steps: 1) fresh, unfixed tissues are frozen rapidly in isopentane quenched in a liquid nitrogen bath; 2) cryostat sections are cut, removed from the knife with a room temperature slide, and then air dried for 5-10 minutes; 3) slides can be stained directly with picro-Ponceau or toluidine blue procedures or with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-CaCl₂ (1.25%). For analysis of mature rat adipose depots steps 2 and 3 are modified as follows: 2) cryostat sections are removed from the knife with a cold slide (-20 C) and dried for 30 minutes at 4 C; 3) the mounted sections are stained with oil red O following fixation for 30 minutes in cold (4 C) 10% formalin-HgCl₂ (2.5%). When procedures described above for immature adipose depots are combined with esterase fining, adipocyte cytoplasm is clearly demonstrated. These procedures allow the routine use of fresh frozen, unfixed cryostat sections in studies of adipose cellularity.