Vinylpyrrolidone-Vinyl Acetate Copolymers as Mounting Media for Azo and Other Dyes

Vinylpyrrolidone-vinyl acetate copolymers (Antara Chemicals, General Aniline and Film Corp., New York N. Y., PVP/VA, E-735 and E-635) were employed as mounting media by adition of ethanol-water to the concentrated ethanolic solution of these plastics (25% plastic in 50% ethanol). The E-735 copolymer was frequently employed and is specifically recommended because it exhibits the highest degree of water tolerance. This type of mounting medium was found to be especially satisfactory in the preservation of azo, oxidation, and other histochemically derived dyes. The medium is useful also in the preservation of stains for fat and certain metachromatic dyes. The inexpensive nature of this mountant and its ease of application recommend it as a useful substitute for glycerol-gelatin.     

Mounting Media for phase Microscope Specimens

-Results with phase microscopy can often be improved by the proper selection of mounting media. Temporary mounts can first be made in liquid mixtures of water and glycerol, butyl carbitol and alpha-chloronaphthalene, alpha-chloronaphthalene and alpha-bromonaphthalene or alpha-bromonaphthalene with methylene iodide to determine what index liquid serves best to emphasize structures of primary interest. Once this has been determined, permanent mounts can be prepared by the substitution of a solid mounting medium that most closely approaches the refractive index of the liquid.     

Combined Fixing, Staining and Mounting Media

A number of non-volatile, water-soluble substances can be added to the usual aceto-cannine fixing fluids. These inert substances do not alter the fixation image and serve as mounting media when the volatile ingredients of the mixture evaporate. Formulae are given for solutions containing dextrin, dextrose, gelatin, pectin, sorbitol, and sucrose. Gum arabic can be incorporated in a formic-acid-carmine fixative. The limiting factor in the use of such mounting media in fixing fluids is the osmotic value they give the solution; with certain precautions, however, they can be used in place of the usual aceto-carmine treatment. The indices of refraction of these media are not as high as those of the natural balsams and the fixation images which the mixtures produce have the characteristic limitations of those secured by the aceto-carmine technic. Some of the natural balsams (Canada balsam, sandarac and Venetion turpentine) can also be incorporated in fixing fluids. These fixatives are able to hold balsam and water in solution together, and, as the volatile components of the mixtures evaporate, the fixed specimens remain in permanent balsam mounts. The addition of carmine to these fluids enables us to fix, stain, dehydrate, clear and mount a specimen in a single operation. These fixatives preserve more details of chromosome structure than the aceto-carmiae fluids, but their use is more limited; and they can be substituted for the latter only with certain favorable material, e.g., pollen mother cells of Rhoeo and Tradescantia and salivary gland chromosomes of Cbirono-mus. Some of the newer synthetic resins can be substituted for the natural balsams. Formulae are given for fixatives which contain Venetian turpentine, sandarac, Canada balsam and two synthetic resins.     

Molecular Model for the Solubilization of Membranes into Nanodisks by Styrene Maleic Acid Copolymers

A recent discovery in membrane research is the ability of styrene-maleic acid (SMA) copolymers to solubilize membranes in the form of nanodisks allowing extraction and purification of membrane proteins from their native environment in a single detergent-free step. This has important implications for membrane research because it allows isolation as well as characterization of proteins and lipids in a near-native environment. Here, we aimed to unravel the molecular mode of action of SMA copolymers by performing systematic studies using model membranes of varying compositions and employing complementary biophysical approaches. We found that the SMA copolymer is a highly efficient membrane-solubilizing agent and that lipid bilayer properties such as fluidity, thickness, lateral pressure profile, and charge density all play distinct roles in the kinetics of solubilization. More specifically, relatively thin membranes, decreased lateral chain pressure, low charge density at the membrane surface, and increased salt concentration promote the speed and yield of vesicle solubilization. Experiments using a native membrane lipid extract showed that the SMA copolymer does not discriminate between different lipids and thus retains the native lipid composition in the solubilized particles. A model is proposed for the mode of action of SMA copolymers in which membrane solubilization is mainly driven by the hydrophobic effect and is further favored by physical properties of the polymer such as its relatively small cross-sectional area and rigid pendant groups. These results may be helpful for development of novel applications for this new type of solubilizing agent, and for optimization of the SMA technology for solubilization of the wide variety of cell membranes found in nature.     

Interim Report of the Committee on Histologic Mounting Media: Resinous Media

The Fisher “Permount” naphthalene polymer, the Hartman Leddon “H.S.R.” terpene polymer resin, a Monsanto polystyrene P-1, the Will Corporation “Diaphane” and “Green Diaphane”, and the du Pont “Lucite” methyl methacrylate polymer were examined, and the possibility of use of some other plastics was also explored. The first 5 mentioned were tested for color preservation of a variety of stains in comparison with Canada balsam and Clarite X. From this point of view polystyrene, the Hartman Leddon “H.S.R.” and the Fisher “Permount” resins were the most satisfactory, then the “Diaphanes”. Both “Permount” and “H.S.R.” show some yellowing. The H.S.R. with a melting point of 115°C, the Permount with 150°C. melting point, and the Polystyrene with a thermal denaturation point above 220°C. all excell Canada balsam in heat resistance. Trimethylbenzene, cymene and monoamylbenzene appear to be the best solvents for polystyrene. Mounts made in a solution of 20 g. polystyrene in 100 ml. trimethylbenzene can be packed flat slide to slide in 24 hours after mounting without sticking together.

This report is not intended to deprecate the use of other resinous mounting media which have not as yet been tested or compared with those mentioned herein.     

Chloroperoxidase-catalyzed Epoxidation of Styrene in Aqueous and Nonaqueous Media

To overcome poor product yields and stability in aqueous solution, we have examined the chloroperoxidase (CPO from Caldariomyces fumago ) catalyzed oxidation of styrene in organic media using tert -butyl hydroperoxide as external oxidant. CPO's intrinsic catalytic activity in tert -butanol, as reflected in its k cat value, was ca. one-fourth of that in aqueous buffer, indicating that the enzyme remains highly active in the organic solvent. Styrene epoxidation reactions were modeled in both aqueous and nonaqueous media to provide global kinetic information, which dominates non-initial rate conditions and is heavily influenced by continuous deactivation of the CPO. Deactivation studies revealed that the enzyme is deactivated quickly by the combination of the tert -butyl hydroperoxide and styrene, possibly due to the styrenic free radicals generated during the enzymatic reaction. These results may enable catalyst-engineering strategies to be initiated to improve the prospects of using CPO in nonaqueous media for large-scale epoxidation reactions.     

Chloroperoxidase-catalyzed Epoxidation of Styrene in Aqueous and Nonaqueous Media

To overcome poor product yields and stability in aqueous solution, we have examined the chloroperoxidase (CPO from Caldariomyces fumago ) catalyzed oxidation of styrene in organic media using tert -butyl hydroperoxide as external oxidant. CPO's intrinsic catalytic activity in tert -butanol , as reflected in its k cat value, was ca. one-fourth of that in aqueous buffer, indicating that the enzyme remains highly active in the organic solvent. Styrene epoxidation reactions were modeled in both aqueous and nonaqueous media to provide global kinetic information, which dominates non-initial rate conditions and is heavily influenced by continuous deactivation of the CPO. Deactivation studies revealed that the enzyme is deactivated quickly by the combination of the tert -butyl hydroperoxide and styrene, possibly due to the styrenic free radicals generated during the enzymatic reaction. These results may enable catalyst-engineering strategies to be initiated to improve the prospects of using CPO in nonaqueous media for large-scale epoxidation reactions.     

False Cellular Localization of Aminopeptidase Caused by Resinous Mounting Media

Fresh frozen sections of rat submandibular gland were processed for leucine aminopeptidase localization with L-lencyl-β-naphthylamide hydrochloride and L-leucyl-β-naphthylamide. When the first substrate was utilized stained sections were dehydrated in an ethyl alcohol series, cleared in xylene, and mounted in water-insoluble (resinous) media. Sections were also removed at each stage of dehydration and cleared and mounted with glychrogel. When the second substrate was used, tissues were partially decolorized in 40% ethyl alcohol and mounted directly in glychrogel. Comparison of all sections mounted in glychrogel indicated that there was no variation in cellular localization, regardless of substrate used or degree of dehydration. Nuclei were unstained. After mounting in balsam or synthetic resin the nuclei exhibited an intense stain and the parenchymal reaction was stronger, but diffuse. Progressive staining of the nuclei was observed, microscopically, immediately after applying the resinous mounting medium. The use of an aqueous mounting medium appears to be mandatory in this procedure—glychrogel is recommended.     

Some Synthetic Resins IN Combined Fixing, Staining and Mounting Media

Many of the recently devised plasticizers and resins can be utilized to advantage in cytological technics. Some of them have solubilities which enable us to incorporate them in such fixing and staining solutions as aceto-carmine and propiorric-carmine. They are non-volatile, do not alter the fixation images of the fluids with which they are mixed, and serve as mounting media as the volatile components evaporate. Thus it is possible to make a permanent slide in a single operation. These newer compounds are better adapted for this technic than are the natural balsams which have been used previously, as their greater tolerance for water provides a much greater margin of safety. Procedures are described for the utilization of (1) Rezyl 7020, a water-soluble resin (now, unfortunately, not available), which dries to form a water insoluble film, (2) Amberol 750 and (3) Bakelite BR-7160, two alcohol soluble resins, more miscible in solutions containing water than are the natural balsams. Formaldehyde can be included in the aceto-carmine and propionic-carmine fluids with the result that more nuclear detail is preserved. Lacto-gelatin has some valuable properties as a mounting medium and can be used when the specimen is stained with orcein. Carmine, which gives a permanent stain in Rezyl 7020, Amberol 750 and Bakelite BR-7160 fades in lacto-gelatin.     

Improved Stability of A Purified Glycol Methacrylate Preparation: Comments

The technical note from Bernier et al. (1984) presents additional observations on our procedure for purifying glycol methacrylate (GMA), a hydrophylic resin (Chappard et al. 1982). It is becoming increasingly popular and widely used as an embedding medium for light microscopic studies. GMA is prepared by esterification of methacrylic acid (MA), but about 1% of free unreacted MA remains in the monomer. MA can copolymerize with GMA and it also binds strongly to thiazin and other basic dyes (Tipett and O'Brien 1975) so as an undesirable impurity it must be removed.     

Effects of Mounting Media on Fading of Toluidine Blue and Pyronin G Staining in Epoxy Sections

Available mounting media cause fading of histological preparations over time. A study was designed to find the most suitable medium for durable mounting of Araldite embedded semithin sections of rabbit cerebral cortex stained with toluidine blue and pyronin G. Among four synthetic mounting media tested, only DePeX prevented fading of the sections during the first month. All mounting media tested helped preserve staining intensity after one month, since the fading rate after one year is only about half that in sections prepared without mounting medium. The average optical density of sections after one year was higher in preparations mounted with DePeX than in sections treated with the other mounting techniques tested in this study. After one year, the average optical density of sections mounted with DePeX had decreased approximately 20%.     

Embedding Media for 1-2 Micron Sectioning. 2. Hydroxyethyl Methacrylate Combined with 2-Butoxyethanol

A hydroxyethyl methacrylate monomer medium incorporating 2-butoxyethanol requires 2 stock solutions for embedding. Solution A: 80 ml of hydroxyethyl methacrylate (Rohm and Haas Co., Philadelphia, Pa.) is mixed well with 16 ml of 2-butoxyethanol; 0.27 gm of benzoyl peroxide, the catalyst, is added and permitted to dissolve. Heating to 40-50 C may be used to accelerate its solution. Solution B: polyethylene glycol 200 or 400, 15 parts, and N,N-dimethylaniline, 1 part, are mixed thoroughly. Tissues are dehydrated in the customary manner to absolute ethanol or other comparable dehydrant, infiltrated completely with A, then cast in a mixture consisting of 42 parts of A well mixed with 1 part of B. Polymerizaion occurs in 4-7 hr. In a water bath at 20 C the time required was about 7 hr; at 28 C, 4 hr. This medium is based on the author's water-polyethylene glycol-hydroxyethyl methacrylate monomer medium (Stain Techn., 42: 119-23, 1967).     

Influence of Mounting Media on the Fading of Basic Aniline Dyes in Epoxy Embedded Tissues

A simple cytophotometric technique is used to quantitate stain fading of basic aniline dye-stained epoxy-embedded tissues mounted in six different commonly used mountants. Significant fading was detected with all six mountants, although rates varied. the lowest rate of fading was observed with immersion oil and the highest rate of fading with Canada balsam. No significant differences in fading rates of four synthetic mounting preparations were observed.