Autoradiography of Water-Soluble Materials in Plant Tissues

Samples of tissue with water-soluble substances are lyophilized and doubly-infiltrated with parlodion and paraffin. Sections are mounted on an adhesive-coated cover slip with chloroform. The reverse side of the cover slip is coated successively with a thin layer of Harleco resin and a thick layer of Kodak “Opaque”. A corner of the cover slip is broken off as a marker. The cover slip is assembled with a covering glass slide on a nuclear track plate (Kodak NTB-2) with protective coating, the section being in contact with the emulsion, and held in place during radioactive exposure. Before development, the cover slip and plate are exposed briefly to light and then disassembled. Following development of the plate and removal of the layer of resin and opaque from the cover slip, staining of the section is optional. Lining up of the section with its autoradiograph can be accurate within 1-5μ.     

Autoradiography of Water-Soluble Materials in Plant Tissues

Samples of tissue with water-soluble substances are lyophilized and doubly-infiltrated with parlodion and paraffin. Sections are mounted on an adhesive-coated cover slip with chloroform. The reverse side of the cover slip is coated successively with a thin layer of Harleco resin and a thick layer of Kodak “Opaque”. A corner of the cover slip is broken off as a marker. The cover slip is assembled with a covering glass slide on a nuclear track plate (Kodak NTB-2) with protective coating, the section being in contact with the emulsion, and held in place during radioactive exposure. Before development, the cover slip and plate are exposed briefly to light and then disassembled. Following development of the plate and removal of the layer of resin and opaque from the cover slip, staining of the section is optional. Lining up of the section with its autoradiograph can be accurate within 1-5μ.     

The Membrane Method of Electron Microscope Autoradiography

Thin sections of methacrylate and Araldite embedded tissues labelled with radioactive isotopes were transferred with a wire loop or brush from the knife edge onto thin formvar membranes which covered 7 mm holes in 76 × 25 × 1.5 mm or 76 × 38 × 1.5 mm plastic slides. To facilitate the mounting of sections, a platform supported the plastic slides close to the ultramicrotome knife. Photographic emulsion diluted 1:5 or 1:10 with water was applied with a pipette to the upper surface of each formvar membrane to cover the mounted sections. Excess emulsion was drained off and the remaining thin film was dried on a warm plate at 45 C to produce a uniform layer over the sections. After storing in the dark for several weeks, preparations were processed in photographic solutions and washed, and sometimes stained, before applying electron microscope grids to the underside of each formvar membrane. To detach each grid with its adherent formvar, section and emulsion, the membrane was pierced around the perimeter of the grid. Grain counts made over nuclei of cells labelled with tritiated thymidine indicate that emulsion is uniformly distributed over each section and that quantitative comparison is possible between labelled areas.     

Processing Stripping Film Autoradiographs of Citrus Buds; Avoidance of Entrapped Air between Tissues and Developed Emulsion

Autoradiography was used on paraffin sections for investigating DNA synthesis in vegetative and flower buds of Citrus sinensis (L.) Osbeck. We preferred stripping film to liquid emulsion to obtain a more uniform thickness of the silver grain layer, as this would not vary more than ±10%. Furthermore, Baserga and Nemeroff (1962) have observed a lower sensitivity of fluid emulsion in comparison to stripping film together with occasionally erratic grain count and a slightly higher incidence of mechanical fogging. However, on using the customary procedure it was found that close contact between the surface of the section and the developed emulsion was not maintained when dehydration and clearing was done in the usual manner. The following modifications were therefore introduced to prevent the formation of air pockets between these two layers.     

Processing Stripping Film Autoradiographs of Citrus Buds; Avoidance of Entrapped Air between Tissues and Developed Emulsion

Autoradiography was used on paraffin sections for investigating DNA synthesis in vegetative and flower buds of Citrus sinensis (L.) Osbeck. We preferred stripping film to liquid emulsion to obtain a more uniform thickness of the silver grain layer, as this would not vary more than ±10%. Furthermore, Baserga and Nemeroff (1962) have observed a lower sensitivity of fluid emulsion in comparison to stripping film together with occasionally erratic grain count and a slightly higher incidence of mechanical fogging. However, on using the customary procedure it was found that close contact between the surface of the section and the developed emulsion was not maintained when dehydration and clearing was done in the usual manner. The following modifications were therefore introduced to prevent the formation of air pockets between these two layers.     

A new tool based on two micromanipulators facilitates the handling of ultrathin cryosection ribbons

A close to native structure of bulk biological specimens can be imaged by cryo-electron microscopy of vitreous sections (CEMOVIS). In some cases structural information can be combined with X-ray data leading to atomic resolution in situ. However, CEMOVIS is not routinely used. The two critical steps consist of producing a frozen section ribbon of a few millimeters in length and transferring the ribbon onto an electron microscopy grid. During these steps, the first sections of the ribbon are wrapped around an eyelash (unwrapping is frequent). When a ribbon is sufficiently attached to the eyelash, the operator must guide the nascent ribbon. Steady hands are required. Shaking or overstretching may break the ribbon. In turn, the ribbon immediately wraps around itself or flies away and thereby becomes unusable. Micromanipulators for eyelashes and grids as well as ionizers to attach section ribbons to grids were proposed. The rate of successful ribbon collection, however, remained low for most operators. Here we present a setup composed of two micromanipulators. One of the micromanipulators guides an electrically conductive fiber to which the ribbon sticks with unprecedented efficiency in comparison to a not conductive eyelash. The second micromanipulator positions the grid beneath the newly formed section ribbon and with the help of an ionizer the ribbon is attached to the grid. Although manipulations are greatly facilitated, sectioning artifacts remain but the likelihood to investigate high quality sections is significantly increased due to the large number of sections that can be produced with the reported tool.     

An Adhesive Transfer Method of Cutting and Mounting thin Sections for Autoradiography

A short strip of adhesive transfer cellophane tape is applied to the face of the block to provide backing for the section during cutting and handling. The tape is sealed to the nuclear track plate with the section against the dry emulsion. After exposure the cellophane backing is removed by immersion in water, and the adhesive is dissolved from the section in unleaded gasoline. The section is hydrated and photographic and histological processing are carried out in the usual manner.     

The problem of proprietary dyes,with special reference to alamar blue,a proprietary dye revealed

A short strip of adhesive transfer cellophane tape is applied to the face of the block to provide backing for the section during cutting and handling. The tape is sealed to the nuclear track plate with the section against the dry emulsion. After exposure the cellophane backing is removed by immersion in water, and the adhesive is dissolved from the section in unleaded gasoline. The section is hydrated and photographic and histological processing are carried out in the usual manner.     

Floating-Out Technics for Rapid Placement of Ribbons of Serial Sections on Slides

The floating-out technic, popular for single paraffin sections, can be applied successfully to ribbons of serials by either of two procedures. (1) If spreading time for the sections is uncritical suitable lengths of ribbon for attachment to a slide are laid on water at a temperature about 8° C below the melting point of the paraffin and manipulated with a rubber bulb pipette to form a unit. This ensemble can then be picked up by the slide in much the same manner as a single section. (2) If spreading time is critical, as for objects that have had guide limes embedded with them, several ribbons are arranged on a cold, dry slide and transferred to the water as a unit. Placing the ribbons on the cold slide so that they slightly overhang one end and the sides of the slide allows them to make proper contact with the water as the slide is immersed. To facilitate controllable spreading in both methods, the water should have added to it 0.5 ml of albumen-glycerol adhesive per 100 ml. Adding water to the slide after the sections have been picked up or manipulation of the ribbons is generally unnecessary if the ribbons have been aligned accurately on the floating-out bath.     

Serial Sectioning of Tissues in Glycol Methacrylate by Supplementary Embedding in a Paraffin-Plastic Matrix

Glycol methacrylate, while offering certain advantages over paraffin as an embedding medium, is difficult to use because it will not ribbon. Rohde (1965) developed a method for producing ribbons of methacrylate sections, but we had little success with it because the ribbons tended to fall apart when even slight stresses were applied to them. We have therefore made use of the principle of double embedding, as this has been used for obtaining serial sections of material embedded in nitrocellulose.     

Serial Sectioning of Tissues in Glycol Methacrylate by Supplementary Embedding in a Paraffin-Plastic Matrix

Glycol methacrylate, while offering certain advantages over paraffin as an embedding medium, is difficult to use because it will not ribbon. Rohde (1965) developed a method for producing ribbons of methacrylate sections, but we had little success with it because the ribbons tended to fall apart when even slight stresses were applied to them. We have therefore made use of the principle of double embedding, as this has been used for obtaining serial sections of material embedded in nitrocellulose.     

Combined Carbowax-Paraffin Technic for Microsectioning of Fixed Tissues

A combined Carbowax-paraffin technic for microsectioning fixed tissues gave ribbon sections as do paraffin infiltrated and embedded tissues. Blocks of formalin or alcohol fixed tissues 2 mm. thick were infiltrated with H.E.M. (Polyethlene Glycol: Carbowax, Hartman-Leddon Co.) or with one of the following polyethylene glycol ester waxes (Glycol Products Co., Inc.) for 4 hours at (61°C.): Polyethylene Glycol 600(Di) Stearate; Carbowax 1000-(Mono) Stearate; Carbowax 4000 (Mono) Stearate; Carbowax 4000 (Mono) Laurate; Carbowax 6000 (Mono) Oleate. The Carbowax infiltrated tissues were placed for 10 minutes in xylene (61° C.), into paraffin (61° C.) for 30 minutes, then into molten paraffin contained in separate molds. (The xylene passage can be excluded for preparations which preclude its use). The blocks were hardened rapidly by submerging in ice water and were fastened to carriers as in the usual paraffin technic. Tissues were cut 6 µ thick. Segments of ribbon were spread on a water bath and mounted on slides. After drying, tissues were stained directly with hematoxylin-eosin or were carried through xylene and alcohols as in routine paraffin preparations prior to staining. The Sudan III fat stain and Best's carmine stain for glycogen were applied as in usual technics. Cellular detail was well preserved and structures did not show the extent of distortion and shrinkage encountered in ordinary paraffin technic preparations.     

Synthesis of Novel Plant Growth Inhibitors Structurally Related to Abscisic Acid

When paraffin wax is dispersed in medium as emulsion, some kinds of bacteria and yeasts readily grow on it. This paper presents a study on microbial cell production from solid paraffin. In this study a paraffin wax which contains 91% of normal paraffins ranging from C₂₅ to C₃₇ with the melting point of 62.5°C was used as a substate, but no solvent was used for the dispersion of the wax.

As a result of this study, the following have been found out. (1) Many strains of liquid normal paraffin assimilating bacteria and yeasts can assimilate paraffin wax. (2) Dried cell yields on added hydrocarbons of Corynebacterium hydrocarboclastus S-12-B2 and Candida tropicalis S-315-Y1 are 70% and 56% respectively, when they are cultured by wax emulsion of 0.6% concentration. (3) When nonion surface-active agent (Plysurf A210G) was added as an emulsifing agent, highly concentrated wax emulsion was obtained, but the growth of microorganisms on it was slower. Further investigation is needed to obtain better strains of bacteria and yeasts and also to find out optimum culture conditions.     

A New Method for Reducing Static Electricity During Paraffin Sectioning

Static electricity interferes with the production of good ribbons of thin paraffin sections. Sections tend to stick to the knife leading to compression, shredding and paraffin sections. Sections ribbon disintegration. the static electricity that builds up is caused by friction between the knife and the tissue block and by the rubbing together of the operator's clothing and sectioning table (Mattheij and Dignum 1975, Bryan and Hughes 1976).     

Double Embedding Broad Thin Leaves in Paraffin

Paraffin pellets were melted in 24 × 24 × 5 mm stainless steel base molds. Specimens of leaves, 18 × 18 mm, were fixed, dehydrated and infiltrated with paraffin. Two specimens were transferred into molten paraffin on their laminar surfaces in a base mold and moved quickly onto a cold surface to cast them in a shallow block of paraffin. Each block was then scored with a razor blade, broken into two primary blocks, and trimmed to 20 × 9 mm with 5 mm flat edges. Each primary block was immersed upright on its long edge in a 22 × 22 × 20 mm Peel-A-Way® embedding mold containing molten paraffin. The leaf edge was held centrally in the mold while moving the double embedment onto a cold surface. In this secondary block, the leaf specimen stood perpendicular to the sectioning surface in perfect orientation for transverse ribbon sectioning. The two phases of paraffin bonded well.     

A New Method for Reducing Static Electricity During Paraffin Sectioning

Static electricity interferes with the production of good ribbons of thin paraffin sections. Sections tend to stick to the knife leading to compression, shredding and paraffin sections. Sections ribbon disintegration. the static electricity that builds up is caused by friction between the knife and the tissue block and by the rubbing together of the operator's clothing and sectioning table (Mattheij and Dignum 1975, Bryan and Hughes 1976).     

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.     

Preparation and Thermal Energy Storage of Carboxymethyl Cellulose-Modified Nanocapsules

A series of nanocapsules with carboxymethyl cellulose (CMC)-modified melamine-formaldehyde as the shell material and phase change paraffin as the core material were prepared by in situ polymerization. The modified capsules were examined using Fourier transform infrared spectra, scanning electronic microscope, differential scanning calorimeter, and optical microscopy, and two factors that influence paraffin emulsion preparation (emulsifier type and stirring rate) were investigated. The effects of the synthesis conditions used for the prepolymer on the surface morphology of the capsules were also studied. We found that phase change capsules prepared with both anionic and nonionic emulsifiers were superior to those prepared with a simple emulsifier. The best performance of the paraffin emulsion was obtained when the emulsion was stirred at 8,000 rpm during preparation. The optimal prepolymer reaction conditions to give smooth capsules with good dispersibility and complete morphology were reaction temperature 72.5 °C, reaction time 75 min, and pH?8.5. The CMC-modified nanocapsules have a phase change enthalpy of 83.46 J/g, are fully encased, and are uniform, with an average particle size of 50 nm.