Pasternack's Paraffin Method Modified for Plant Tissue

This method for preparing paraffin sections of plant material is a modification of Pasternack's one-hour method for animal tissues. Fixation in Randolph's CRAF fixative is hastened by heat and increased vapor pressure obtained by the use of screw top vials. Dehydration with Zirkle's butyl alcohol series likewise is hastened in the same manner. The rapid penetration of paraffin by the use of 1/2 paraffin and 1/2 butyl alcohol in heated screw top vials shortens the embedding process. Sections are held on the slide thru staining by albumen fixative and a coating of 0.2% celloidin in absolute alcohol and ether. Good penetration with freedom from shrinkage or distortion is obtained and root tip chromosome counts can be made in approximately 3 hours.     

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.     

Paraffin Embedding; a Graded-Temperature Table Used with Multiple Blocking for Enhanced Efficiency

Details are given for the construction of a graded-temperature table having a cool and hot side. Aluminum baking pans smeared with glycerol are used to cast the multiple paraffin blocks. These are loaded with paraffin wax from an electrically heated paraffin wax dispenser incorporated in the graded-temperature table and the pieces of tissue and labels orientated in them. The pans are then moved to the cool side of the table before finally floating them on cold water to harden the wax. Arranged above the heated side of the table are two infrared lamps which prevent premature solidification of the surface of the wax. After removing the solid, multiple-block slabs from the pans, the infrared lamps are used to soften the wax to a cheese-like consistency. Individual blocks are cut apart and trimmed around their sides with single strokes to produce smooth-sided blocks which are ready for attaching to wooden holders, adapted to the microtome chuck.     

A Disposable Plastic Box for Paraffin Embedding

A sheet of cellulose acetate about 0.01 inch thick is clamped over a mold, heated to softness by an electric heater and drawn down over the mold by means of a vacuum. When cooled, the sheet, now formed into embedding boxes, is removed from the clamp. Boxes so made are inexpensive enough to be disposable but can be reused, since the sides of the boxes are sloped to allow easy removal of the paraffin block.     

Letters to the Editor

Abstract

Hot commercial dishwashing detergent has been used to deparaffinize and hydrate formalin fixed, paraffin embedded sections for immunohistochemistry. Fifty-five antibodies, used routinely for diagnosis, were used to compare hot detergent dewaxing with the proprietary hydrocarbon-based dewaxing reagent supplied with the Bond Max immunohistochemistry system^®. A 2% concentration of commercial dishwashing detergent in distilled water was heated to 90° C and paraffin sections were treated twice for 1 min each. Nearly all antibodies gave equivalent results except CD10 and CD57 (hydrocarbon-based dewaxing better) and CD45 and alpha fetoprotein (detergent dewaxing better); the differences, however, were minimal. There also was a significant cost saving using detergent dewaxing.     

Acceleration of the Movat Pentachrome One Stain by Heat

The conventional staining time for Movat's pentachrome I stain (Arch. Path., 60: 289-295, 1955) was shortened from about 18-19 hr to about 2.5 hr. The ammoniated alcohol and the resorcin-fuchsin staining baths were heated to 56 C. All other steps in the technic were performed at 25-27 C. Staining properties of paraffin sections of many types of tissue fixed in formalin, formol-sublimate-acetic, or in Bouin's fluid, showed that staining with resorcin-fuchsin at the elevated temperature gave the same results as staining at room temperature.     

Characterization of Fast-Scan Cyclic Voltammetric Electrodes Using Paraffin as an Effective Sealant with In Vitro and In Vivo Applications

Fast-scan cyclic voltammetry (FSCV) is a powerful technique for measuring sub-second changes in neurotransmitter levels. A great time-limiting factor in the use of FSCV is the production of high-quality recording electrodes; common recording electrodes consist of cylindrical carbon fiber encased in borosilicate glass. When the borosilicate is heated and pulled, the molten glass ideally forms a tight seal around the carbon fiber cylinder. It is often difficult, however, to guarantee a perfect seal between the glass and carbon. Indeed, much of the time spent creating electrodes is in an effort to find a good seal. Even though epoxy resins can be useful in this regard, they are irreversible (seals are permanent), wasteful (epoxy cannot be reused once hardener is added), hazardous (hardeners are often caustic), and require curing. Herein we characterize paraffin as an electrode sealant for FSCV microelectrodes. Paraffin boasts the advantages of near-immediate curing times, simplicity in use, long shelf-life and stable waterproof seals capable of withstanding extended cycling. Borosilicate electrode tips were left intact or broken and dipped in paraffin embedding wax. Excess wax was removed from the carbon surface with xyelenes or by repeated cycling at an extended waveform (-0.4 to 1.4V, 400 V/s, 60 Hz). Then, the waveform was switched to a standard waveform (-0.4 to 1.3V, 400 V/s, 10 Hz) and cycled until stable. Wax-sealing does not inhibit electrode sensitivity, as electrodes detected linear changes in dopamine before and after wax (then xylenes) exposure. Paraffin seals are intact after 11 days of implantation in the mouse, and still capable of measuring transient changes in in vivo dopamine. From this it is clear that paraffin wax is an effective sealant for FSCV electrodes that provides a convenient substitute to epoxy sealants.     

A new method for easy demonstration of argyrophil cells.

A new method for silver impregnation of endocrine cells of the gastrointestinal mucosa is described. It offers great reliability, eveness of impregnation, and, since it can be used on batches of slides, is also suitable for histology class and investigation material. The procedure for paraffin sections of formalin-fixed material is as follows: dewax and transfer to distilled water, leave in 0.5% silver nitrate solution for 2 hours at 60 C. Rinse in distilled water, then treat in Bodian developer (hydroquinone, 1 g; sodium sulphite, 5 g; distilled water, 100 ml) previously heated to 60 C. Rinse in running tap water, distilled water, and then re-impregnate for 10 minutes at 60 C in the same silver solution and reduce in Bodian's solution. Since the background is not impregnated by this method, sections may be counterstained by any basic anilin dye to bring out nuclei. A 0.1% kernechtrot solution was found very satisfactory in this respect. The granulations of argyrophil cells stand out sharply black against a red background.     

A New Method for Easy Demonstration of Argyrophil Cells

A new method for silver impregnation of endocrine cells of the gastrointestinal mucosa is described. It offers great reliability, eveness of impregnation, and, since it can be used on batches of slides, is also suitable for histology class and investigation material. The procedure for paraffin sections of formalin-fixed material is as follows: dewax and transfer to distilled water, leave in 0.5% silver nitrate solution for 2 hours at 60 C. Rinse in distilled water, then treat in Bodian developer (hydroquinone, 1 g; sodium sulphite, 5 g; distilled water, 100 ml) previously heated to 60 C. Rinse in running tap water, distilled water, and then re-impregnate for 10 minutes at 60 C in the same silver solution and reduoc in Bodian's solution. Sma the background is not impregnated by this method, sections may be counterstained by any basic anilin dye to bring out nuclei. A 0.1% kernechtrot solution was found very satisfactory in this respect. The granulations of argyrophil cells stand out sharply black against a red background.     

Some Responses of Tsetse Flies to Visual and Olfactory Stimuli

HISTOCHEMICAL fluorescence experiments, using the modifications^1,2 of the formaldehyde condensation procedure described by Falck et al.³, are increasingly common. The procedures for demonstrating monoamines in freeze-dried tissue, require that the sections cut from formaldehyde gas-treated tissue be kept away from water and so the conventional heated water bath for relaxing the cut paraffin sections cannot be used. Acetonitrile⁴ and liquid paraffin⁵ have been utilized and some laboratories even use a heated mercury bath (S. Norr, personal communication), although what is desired is a very non-reactive liquid. ‘Fluorinert Electronic Liquids FC-75, FC-77’ (3M Company, St Paul, Minnesota 55101) adequately replace the water bath. At 45° C these two clear liquids allow cut sections to be handled in the same manner as in the conventional water bath. Other liquids in this series can probably be chosen to give similar characteristics at other bath temperatures, although they have not been tried. A bath with intermediate qualities could be made as the liquids in the series are completely miscible with one another. The ‘Fluorinert Electronic Liquids’ are stated by their manufacturer to be very non-reactive and to have very low water and oil solubilities. The inertness of these liquids suggests that they may also be used to float sections as they are cut and to immerse freeze-dried tissue for storage.     

Polystyrene Embedding: A New Method for Light and Electron Microscopy

Polystyrene embedments of histological specimens can be Obtained with a solution 1 : 4 polystyrene-toluene, 5% benzyl alcohol and 1% dibutyl phthalate, allowing the solvent to evaporate in polyethylene containers for 2-3 days at 58 C. The resulting blocks are easily cut into truncated pyramids, each containing a piece of tissue. which are then glued to a Plexiglas support Drying is completed at 80 C for 20 hr. The pyramids can then be sectioned to produce thick sections, with a steel knife or to produce semi- or ultrathin sections with a glass knife. A 10% paraldehyde solution is used to mount the light microscopy dons on a slide heated on a hot plate to 80 C; those can be treated with the same techniques used with paraffin sections. The results are of high quality. Semithin sections of tissues fired for electron microscopy can be stained directly after mounting, or by a wider range of stains once the polystyrene has been removed by organic solvents. In electron-microscopy, the ultrathin sections obtained with the usual techniques are highly electron beam-resistant and give acceptable results.     

Polystyrene embedding: a new method for light and electron microscopy.

Polystyrene embedments of histological specimens can be obtained with a solution of 1:14 polystyrene-toluene, 5% benzyl alcohol and 1% dibutyl phthalate, allowing the solvent to evaporate in polyethylene containers for 2-3 days at 58 C. The resulting blocks are easily cut into truncated pyramids, each containing a piece of tissue, which are then glued to a Plexiglas support. Drying is completed at 80 C for 20 hr. The pyramids can then be sectioned to produce thick sections with a steel knife or to produce semi- or ultrathin sections with a glass knife. A 10% paraldehyde solution is used to mount the light microscopy sections on a slide heated on a hot plate to 80 C; these can be treated with the same techniques used with paraffin sections. The results are of high quality. Semithin sections of tissues fixed for electron microscopy can be stained directly after mounting, or by a wider range of stains once the polystyrene has been removed by organic solvents. In electron microscopy, the ultrathin sections obtained with the usual techniques are highly electron beam-resistant and given acceptable results.     

DNA extraction from archival formalin-fixed, paraffin-embedded tissue sections based on the antigen retrieval principle: heating under the influence of pH.

During the course of diagnostic surgical pathology, pathologists have established a large collection of formalin-fixed, paraffin-embedded tissues that form invaluable resources for translational studies of cancer and a variety of other diseases. Accessibility of macromolecules in the fixed tissue specimens is a critical issue as exemplified by heat-induced antigen retrieval (AR) immunohistochemical (IHC) staining. On the basis of observations that heating may also enhance in situ hybridization (ISH) and the similarity of formalin-induced chemical modifications that occur in protein and in DNA, we designed a study to examine the efficiency of DNA extraction from archival formalin-fixed, paraffin-embedded tissues using an adaptation of the basic principles of the AR technique, i.e., heating the tissue under the influence of different pH values. Archival paraffin blocks of lymph nodes, tonsil, and colon were randomly selected. Each paraffin block was prepared in 34 microtubes. For each paraffin block, one tube was used as a control sample, using a non-heating DNA extraction protocol. The other 33 tubes were tested using a heating protocol under 11 variable pH values (pH 2 to 12) under three different heating conditions (80, 100, and 120C). Evaluation of the results of DNA extraction was carried out by measuring yields by photometry and PCR amplification, as well as kinetic thermocycling (KTC)-PCR methods. In general, lower pH (acid) solutions gave inferior results to solutions at higher pH (alkaline). Heating tissues at a higher temperature and at pH 6-9 gave higher yields of DNA. There appeared to be a peak in terms of highest efficiency of extracted DNA at around pH 9. The average ratios 260:280 of extracted DNA also showed better values for samples heated at 120C. PCR products of three primers showed satisfactory results for DNA extracted from archival paraffin-embedded tissues by heating protocols at pH 6-12, with results that were comparable to the control sample subjected to the standard non-heating, enzymatic DNA extraction method. This study is the first to document the use of heating at an alkaline pH for DNA extraction from archival formalin-fixed, paraffin-embedded tissues, a recommendation based on the principles of AR for protein IHC. These findings may lead to a more effective protocol for DNA extraction from archival paraffin-embedded tissues and may also provide enhanced understanding of changes that occur during formalin-induced modification of nucleic acids.     

N-butyl Methacrylate and Paraffin as an Embedding Medium for Light Microscopy

A method of tissue embedding using n-butyl methacrylate and paraffin is described. Following alcohol dehydration and infiltration with the methacrylate monomer, tissues are embedded in gelatin capsules in a mixture consisting of 3.5 g of paraffin for each 10 ml of methacrylate. Benzoyl peroxide (0.2 g for each 10 ml of monomer) is added as the catalyst and the methacrylate polymerized in a 50 C oven for 18-24 h. Following polymerization the block is trimmed and embedded in paraffin to provide a firm support during sectioning. A water trough attached to the microtome knife is essential to facilitate the handling of sections and ribbons. For serial sections a mixture of equal weights of beeswax and paraffin is used to make the sections adhere to each other. Usual staining procedures can be used since the embedding medium is readily soluble in xylene.     

N-Butyl methacrylate and paraffin as an embedding medium for light microscopy.

A method of tissue embedding using n-butyl methacrylate and paraffin is described. Following alcohol dehydration and infiltration with the methacrylate monomer, tissues are embedded in gelatin capsules in a mixture consisting of 3.5 g of paraffin for each 10 ml of methacrylate. Benzoyl peroxide (0.2 g for each 10 ml of monomer) is added as the catalyst and the methacrylate polymerized in a 50 C oven for 18--24 h. Following polymerization the block is trimmed and embedded in paraffin to provide a firm support during sectioning. A water trough attached to the microtome knife is essential to facilitate the handling of sections and ribbons. For serial sections a mixture of equal weights of beeswax and paraffin is used to make the sections adhere to each other. Usual staining procedures can be used since the embedding medium is readily soluble in xylene.     

Piccolyte Investments for Better Section Cutting

Piccolyte 115 (β-pinene polymers) added to Tissuemat, Paraplast or Peel-Away embedding media is recommended for investment of paraffin infiltrated tissues. Mixed with paraffin at 3% and 10% and used for double embedding of paraffin infiltrated tissues, Piccolyte 115 permits good, complete sections virtually free of folds or wrinkles in less time and with less effort than with paraffin embedding alone.     

Birefringence and Paraffinophilia of Cell Nuclei

Birefringence of cell nuclei was present in most tissues but seen exclusively in paraffin sections. Only after staining with an acridinc derivative (rivanol) was it found in smears and frozen sections. Although retention of paraffin in the nucleus contributes, for the most part to its anisotropy, present evidence supports the hypothesis that the chemical nature and the physical state of nuclear material, especially of the DNA, plays the important role as a substrate, which selectively binds of paraffin molecules. This evidence is based mainly on the blocking effect on birefringence which occurs when small pieces of tissues are treated in toto, before paraffin embedding, with DNA-extracting procedures and nuclear stainings. Moreover, the variability in degree and extent of birefringence noted in different tissues corroborates this view. Factors in the preparatory procedure, i.e, deparaffinization, hydration and dehydration, were found to affect markedly the binding of paraffin to nuclear substance. Nevertheless, if paraffin affinity for nuclei is not considered, it may introduce inaccuracies into methods designed to determine the nuclear mass quantitatively, after staining.     

常规石蜡切片方法的改良

针对传统石蜡切片方法中的缺陷,对制片方法进行了相应的改良。总结了切片制作过程中可能存在的问题以及处理对策;提出了一些能缩短实验周期,解决实验有毒物质二甲苯污染的方案。结合教学实践发现改良方案有助于提高石蜡切片的质量。     

Phenotypic Adaptations Help Rhodococcus erythropolis Cells during the Degradation of Paraffin Wax

During crude oil extraction, the reduction in temperature and pressure results in the precipitation of paraffin wax that contains 20–40 carbon chain hydrocarbons. The paraffin wax may accumulate inside production tubes, pipelines, and processing facilities, and also in tankers during petroleum transportation. There are few bacterial strains that are able to degrade solid substrates. In the present study, the biodegradation of paraffin is evaluated using Rhodococcus erythropolis cells. This bacterium is able to grow using paraffin wax from an oil refinery plant as the sole carbon source. The cells grow as a thick biofilm over the solid substrate, make scale‐like structures that increase the area of the initially smooth surface of paraffin, produce biosurfactants, and become more negatively charged than ethanol‐ or glucose‐grown cells. When paraffin wax is supplied as microparticles, to increase the cell–substrate contact area and to simulate paraffin precipitation, the cells also adjust the composition of the fatty acids of the phospholipids of the cellular membrane to decrease its fluidity and paraffin biodegradation increases considerably. The study suggests that the phenotypic adaptation of R. erythropolis cells may be used to degrade paraffin wax under real conditions.