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.     

Use of a Silicone Rubber (Clear Seal) as a Section Adhesive Resistant to Acid, Alkali and Heat

An optically clear silicone rubber adhesive is recommended for use in histochemical procedures in which detachment of tissue sections is likely. Procedure: Cut paraffin sections and float on a 45-50 C water bath; leave frozen sections on the microtome knife in the cryostat; spread the silicone rubber thinly and evenly over 2/3 of the slide (Clear Seal—General Electric, was used); pick up paraffin sections directly from the floatation water and frozen sections from the microtome knife with a warm slide; dry for 1.5 hr at 25 C; place paraffin sections in a 60 C oven for 0.5 hr, deparaffinize through xylene and hydrate through alcohols to water. Stain sections as desired, but avoid clearing agents before mounting after strong acid or alkaline treatment, and mount rapidly if a synthetic resin is used because of the solvent effect on the silicone rubber. Of the adhesives tried, silicone rubber is the only one capable of withstanding boiling 10% HCl for any period of time without detachment of sections.     

A method for quantitation of protein in the presence of Percoll

An electromagnet was modified for measurements of the magnetic circular dichroism of samples held at cryogenic temperatures using a standard laboratory cryostat. The external dimensions of the cryostat are too great to permit its insertion in the air gap between the poles of the magnet without an unacceptable reduction in the strength of the magnetic field at the sample. This problem was overcome by designing new pole caps which become an integral part of the vacuum system of the cryostat. The ends of the new pole caps project into the body of the cryostat so that the gap between them is 1 in. or less, thus achieving a magnetic field exceeding one Tesla at the sample. No permanent alterations of the cryostat are required. The chief advantages of this design are economy and flexibility since a general purpose cryostat is used instead of a special unit designed to fit in the small space between the poles of an unmodified magnet. The cryostat used in this design cools the sample by conduction; thus the problem of optical distortions resulting from bubbling of liquid nitrogen or other cryogen is avoided and the temperature can be varied continuously using standard auxiliary equipment. Extra windows at 90° with respect to the optical beam permit inspection of the sample in situ and could be used for experiments such as fluorescence-detected magnetic circular dichroism which require optical access perpendicular to the direction of the magnetic field.     

Some new methods for affixing sections to glass slides. III. Pressure-sensitive adhesives

A variety of polymeric formulations have been evaluated for use as pressure-sensitive adhesives in affixing sections of fresh or frozen tissues for histochemistry and other purposes. The best adhesive with respect to water resistance, refractive index, and low temperature tack was polyisobutylene. In mixtures of different molecular weights this adhesive is specially suited for room temperature or cryostat use. Slides coated with this adhesive should only be used with staining or incubation solutions which have been filtered and degassed. Mounting cannot be performed with xylene-containing media, UV-curable monomeric media being favored. For anhydrous processing or nonpermanent preparations, polyethyleneimines, which are soluble in water but insoluble in xylene, may be used. Mixtures of polydimethylsiloxanes can be applied for subsequent temporary mounting with water or aqueous media.     

Influence of fixation and buffer treatment on the release of enzymes from the plasma membrane.

1. Pretreatment of frozon cryostat sections with formaldehyde or calcium ions inhibits diffusion of the plasma membrane enzymes 5' nucleotidase, ATP-ase and alkaline phosphatase during incubation. 2. Treatment of fixed sections with different kinds of buffer at 37 degrees C induces diffusion of enzyme activity from the plasma membrane to other sites of the section and into the incubation medium. This buffer influence depends on temperature: at 4 degrees C only a slight diffusion occurs. Addition of phospholipase C, digitonin or taurocholate to the buffer opposes the buffer effect. 3. Pretreatment of frozen cryostat sections with a mixture of equal parts of chloroform and acetone give a good fixation of the plasma membrane enzymes 5'-nucleotidase, ATP-ase, alkaline phosphate and leucyl-beta-naphthylamidase. During this treatment the different kinds of lipids present in the membrane are ex-racted equally. After this fixation buffer treatment does not cause a visible diffusion of enzyme activity in the section. Only a slight diffusion (1 till 7 percent) into the buffer solution takes place. 4. The mentioned treatments open up possibilities to get insight into the membrane anchorage of plasma membrane enzymes.     

A gas-flow cryostat for use in freeze-quench studies: design and application to discontinuous pre-steady-state spectral analyses

A design for an improved freeze-quench apparatus is presented. A freeze-quench apparatus has two main parts: the apparatus which mixes the reactants and after a set time sprays the reacting mixture into the quencher, and the quenching apparatus itself. The quenching apparatus is the novel feature of our design and it comprises a gas-flow cryostat mounted directly onto a liquid nitrogen storage Dewar. The gas flow maintains the temperature of a small volume of isopentane, which acts as the freeze-quench agent, at -140 degrees C. (The apparatus can maintain the quenching temperature over the range -190 degrees C to room temperature.) Because of the small size of the cryostat and the much reduced volume of solvent used by this method it is more convenient to use than its predecessors, can be used in the open laboratory, and is safer. Our apparatus is designed for application to electron paramagnetic resonance studies but could be easily modified for use with other spectroscopic techniques.     

Glucose-6-phosphate dehydrogenase activity in spinach as measured by image analysis: A new approach for plant enzyme histochemistry

Summary A relatively low-cost computer-assisted image analysis system is described. Software has been specifically written for the continuous monitoring of absorbance readings on cryostat sectons of plant tissues incubated in media to reveal enzyme activities. The equipment was tested by quantify ing glucose-6-phosphate dehydrogenase activity in cryostat sections from shoot apices of spinach plants. The reaction rate of the dehydrogenase activity was monitored at two incubation temperatures, 20°C and 30°C. Control incubations were performed in media lacking substrate. The specific test minus control reaction at 30°C was twice that at 20°C. Variation of the substrate concentration at 30°C yielded a Km value of 0.37 mM. These preliminary results show that our image analysis system can be used for kineti measurements of dehydrogenase activity in froizen tssue sections and constitute a new approach for enzyme histochemistry in the shoot apicla meristem.     

Vacuumless freezing-drying: Its application in catecholamine histochemistry

Summary Details of a simple technique to prepare tissues for the catecholamine reaction are described. Fresh cryostat sections are dried above phosphorous pentoxyde at –25⁰C for 15 hours. Ice evaporates from the sections under normal atmospheric pressure at this temperature. Frozen-dried cryostat sections are treated with formaldehyde gas like other frozendried specimens and observed under the fluorescence microscope immediately. The patterns obtained are at least comparable with those obtained by the original Falck procedure.     

Freeze-Sectioning of Plant Tissues

Techniques are described for freeze-sectioning a wide range of both fresh and fixed plant tissues. Gelatin-antifreeze media are used to support but not infiltrate the tissue during sectioning. At cryostat temperatures of -10 to -15 C, 15% gelatin (w/v) containing 0.8% dimethyl sulfoxide (DMSO), or 1.5% ethanediol (ethylene glycol), or 2% glycerol is used. Lower concentrations of gelatin and higher concentrations of antifreezes are required for sectioning at -24 C. Petri plates of media are stored at 2 C, and used by simply melting a hole in the medium. Fresh tissues can be placed directly in the hole, or prefrozen at temperature of liquid nitrogen, or equilibrated in antifreeze solution, before freeze-sectioning in the gelatin antifreeze medium. Many plant tissues have highly vacuolated cells and need equilibration in antifreeze solutions prior to freeze-sectioning. Fixed tissues are rehydrated and washed in water or buffer for 15-24 hr before equilibrating in a 10% solution of either DMSO, ethanediol or glycerol (named in order of rapidity of equilibration). Pretreatment in 10% DMSO is usually for 1-6 hr at 2 C for histochemical studies; or in 10% ethanediol or glycerol for 15-24 hr at either room temperature or 37 C for morphological studies. These methods permit serial cryostat sections free from freezing and thawing artifacts to be cut as thin as 2 μ.     

The effects of storage on the retention of enzyme activity in cryostat sections. A quantitative histochemical study on rat liver

Summary The effect of storage of unfixed cryostat sections from rat liver for 4 h, 24 h, 3 days and 7 days at -25°C was studied on the activities of lactate dehydrogenase, glucose-6-phosphate dehydrogenase, xanthine oxidoreductase, glutamate dehydrogenase, succinate dehydrogenase (all demonstrated with tetrazolium salt procedures), glucose-6-phosphatase (cerium-diaminobenzidine method), 5-nucleotidase (lead salt method), dipeptidyl peptidase II, acid phosphatase (both simultaneous azo coupling methods), d-amino acid oxidase (cerium-diaminobenzidine-cobalt-hydrogen peroxide procedure) and catalase (diaminobenzidine method). The effect of drying of the cryostat sections at room temperature for 5 and 60 min was investigated as well. The enzyme activities were quantified by cytophotometric measurements of test and control reactions. The test minus control reaction was taken as a measure for specific enzyme activity. It was found that the activities of all the enzymes investigated, with one exception, were affected neither by storage of the cryostat sections at -25°C for up to 7 days, nor by drying of the sections at room temperature for up to 60 min. The exception was xanthine oxidoreductase, whose activity was reduced by 20% after 5 min drying of sections or after 4 h storage. Therefore, only incubations for xanthine oxidoreductase activity have to be performed immediately after cutting cryostat sections, whereas for the other enzymes a considerable margin appears to exist.     

The influence on enzyme activity of storage of tissue blocks at −70° C

Summary Blocks of tissue from various organs of the rat have been chilled by precipitate immersion in n-hexane cooled to –70° C, and then stored at –70° C. At various intervals (up to 14 days) after chilling, cryostat sections were prepared from these blocks and assayed for the activity of a variety of enzymes. Enzyme activity was measured by scanning and integrating microdensitometry. With the exception of acid phosphatase and cytochrome oxidase, all enzymes assayed were stable for at least 7 days after storage at –70° C and most were stable for 14 days. Storage of fresh-frozen sections at –30° C in the cabinet of the cryostat, for up to 24 h, had little effect on enzyme activity.     

Methods for decreasing interstitial immunoglobulin in tissue slices and cryostat sections

To determine whether lymphoid antigens and cellular morphology can be preserved after long-distance transport in buffer or cell culture medium, we stained cryostat sections prepared from human tonsil samples that had been kept at 4 degrees C or 20 degrees C for 24, 48 or 72 h. B-Cell antigens, T-cell antigens, and Ia antigens were well preserved after storage up to 72 h in buffer or medium at 4 degrees C. Interstitial immunoglobulin (Ig) was decreased following all incubation procedures. We then investigated methods to diminish interstitial Ig in cryostat sections, since it would be inconvenient to keep 2-3 mm tissue slices in buffer or medium prior to freezing and subsequent Ig staining. Cryostat sections were air dried or briefly fixed in acetone prior to washing in buffer or medium at 4 degrees C, 20 degrees C or 37 degrees C for 1, 2 or 24 h. Then sections were air dried or washed prior to acetone fixation and immunostaining. A method for washing cryostat sections was developed which diminished interstitial Ig without compromising the quality of immunostaining or cellular detail. These methods are especially useful for studying samples of lymphoid tissue in which the presence of large quantities of interstitial Ig obscures the detection of monotypic Ig staining patterns.     

Immunohistochemical procedures for the demonstration of peptide- and tyrosine hydroxylase-containing nerve fibers in cryostat sections of unfixed rapidly frozen tissue stored for long periods of time. A study on heart tissue

Traditional protocols for the immunohistochemical localization of peptides and tyrosine hydroxylase (TH) in nerve fibers in cryostat sections require the tissue to be thoroughly fixed and rinsed and to be processed for the cryostat sectioning and the immunohistochemical staining more or less directly after freezing. In the present study it was tested whether also unfixed, rapidly frozen tissue, conforming to guinea pig and bovine heart specimens, can be used for the visualization of neuropeptides [neuropeptide Y (NPY) and substance P (S P)] and TH in cryostat sections. The following observations were made: 1) NPY-immunoreactive (IR) and S P-IR nerve fibers could be clearly identified in both fixed and unfixed sections of this type of tissue. 2) TH-IR nerve fibers could be detected in unfixed tissue if the sections were post-fixed with aldehydes by the use of a two-step fixation process related to a sudden change of pH. However, the outlines of the nerve fibers were sometimes diffuse. 3) Storage of unfixed tissue for periods of up to 2.5 years at-80 degrees C did not lead to a decrease in immunoreactivity. 4) Somewhat higher concentrations of primary antibodies had to be used for sections of unfixed tissue than for sections of fixed tissue when the FITC method was used. This waste of antibodies was partly overcome by use of the biotin-streptavidin method. The glyoxylic acid induced catecholamine(CA)-fluorescence method for demonstration of sympathetic nerve fibers was also applied and was found to give optimal results after storage of tissue for up to 2.5 years.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oils as adhesives for seed inoculation and their influence on the survival of Rhizobium spp. and Bradyrhizobium spp. on inoculated seeds

Mineral oil, peanut oil and soybean oil were compared with water and gum arabic for their suitability as adhesives for seed inoculation with peat inoculants. Inoculated seeds were stored at 4, 28 and 34°C, and sampled after 1, 3 and 9 days to determine the survival of rhizobia. Germination and nodulation tests were performed on the inoculated seeds. Results showed that oils were suitable adhesives for peat inoculants. Although the oils initially bound less inoculant to the seed, the number of surviving rhizobia was similar to that obtained by the gum arabic treatment after storage at 28 and 34°C for 3 and 9 days. An interesting finding of this experiment was that peanut and soybean oils were superior to gum arabic in supporting significantly higher numbers of chickpea rhizobia at 34°C. Inoculated seeds tested for germination and nodulation showed no adverse effects from the oil treatments. Oils hold good potential as adhesives for seed application in inoculation technology.H.J. Hoben and P. Somasegaran are with the NIFTAL Project, University of Hawaii, 1000 Holomua Avenue, Paia, HI 96779-9744, USA. Nwe Nwe Aung is with the Institute of Agriculture, Yezin, Pyinmana, Burma. Ui-Gum Kang is with the Yeongnam Crop Experiment Station, Rural Development Administration, P.O. Box 6, Milyang, Korea.     

Staining method for whole-body autoradiography.

Sagittal whole-body sections of frozen mice were cut on a hydraulicly driven microtome in a cryostat at--15 C by applying cotton or nylon-backed adhesive tape to the mouse before cutting. Section thickness was 20 mu. The sections, still adhering to the tape, were dried in the cryostat (-15C) under atmospheric pressure. After autoradiography, the sections were pressed to a glass slide spread with a mixture of albumin and glycerin. The slide was immersed in xylene at 30 C for 15 min. The tape was then removed from the slide, where the section remained to be stained with hematoxylin-eosin. The section thus obtained enabled the tissue histology to be related to the autoradiogram. This method may also be applied to histochemical studies of substances insoluble in xylene.     

Histochemical demonstration of norepinephrine in postmortem tissues

Summary A modified fluorescence method for the demonstration of norepinephrine in cryostat sections may be used with postmortem tissues and surgical specimens stored for as long as 24 hours at 4° C., and 6 hours at room temperature. Quantitative assay for norepinephrine in these tissues show no significant loss of catecholamine during such storage.Supported in part by Grant HE 10465 from the National Institute of Health.     

Attitudes and usage of denture adhesives by complete denture wearers: a survey in Greece and the Netherlands

doi: 10.1111/j.1741‐2358.2011.00566.x
Attitudes and usage of denture adhesives by complete denture wearers: a survey in Greece and the Netherlands Objective: To explore whether there are differences in usage of and attitudes towards denture adhesives among patients in two countries. Background: There are no multi‐country surveys concerning usage of and attitudes towards denture adhesives from complete denture wearers. Materials and methods: The survey took place in Greece and the Netherlands with a sample of 284 and 165 consecutive complete denture wearers, respectively, by using a 9‐item prepared questionnaire. Statistical analysis relied on chi‐square test at α = 0.05. Results: In this survey, 26 and 20% of Greek and Dutch patients, respectively, had tried denture adhesive, but only 27% of them in Greece as well as in the Netherlands currently used it; 49% of the Greek and 45% of the Dutch participants rated the overall performance of adhesives as good. Between the two populations, no differences were identified in a majority of the research variables, except where 27% of Greeks answered that they did not know the existence of denture adhesives compared to none of the Dutch patients and when 90% of the Dutch contrary to 70% of Greeks reported that they did not need denture adhesives as they could manage their dentures well. Conclusion: The usage of and attitudes towards denture adhesives between the Greek and Dutch sample were similar with only two exceptions concerning the knowledge of existence and the need of using denture adhesives.     

An inexpensive alternative to routine section adhesives for histology: the mucilaginous substance of the Assyrian plum

We found that the mucilaginous substance of the Assyrian plum, Cordia myxa, can be used as an adhesive for attaching sections of animal tissues to slides. Unlike Mayer's albumen, this material left no stainable residue and had no noticeable effect on the histological structure of the tissue sections. The mucilaginous substance of C. myxa is a useful and inexpensive alternative to standard adhesives.     

The distribution of 14c from [U-14c]glucose in mice using whole-body autoradiography.

Tissue distribution of radioactive carbon from [U-14C]glucose in the mouse in vivo was studied by whole-body autoradiography. The mice were frozen with Dry-Ice-acetone at 0.5, 1, 5 and 30 min, 1 and 24 hr and 1 and 3 weeks after intraperitoneal injection of [U-14C]glucose. Whole-sagittal sections of the frozen mouse, obtained by using a microtome in a cryostat, were dried in a cryostat and autoradiographed. The resulting dry autoradiographs are called untreated autoradiographs in the present work. The sections were then fixed in cold 6% (w/v) HClO4, dried at room temperature and again autoradiographed. Autoradiographs that have undergone this process are referred to as treated autoradiographs. In both untreated and treated autoradiographs, within 1 min following injection of the labeled glucose, the abdominal cavity had the highest autoradiographic density. At 1 hr, density became highest in Harder's, sublingual and duodenal glands, large intestinal mucosa and tongue, and after 3 weeks, no autoradiographic denisty was present.