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Pickering emulsions stabilized by milled starch particles have been developed as a novel food-grade formulation to enhance the bioaccessibility of poorly soluble bioactive compounds (i.e., curcumin) by controlling the digestion of lipids in the human gastrointestinal (GI) tract. The dynamic bioaccessibilities of curcumin with and without encapsulation in the Pickering emulsion were evaluated using the dynamic TNO's gastrointestinal (TIM-1) model. For comparison, their digestion profiles were also studied using the in vitro pH-stat lipolysis model. With the combination of two in vitro models, the effect of the milled starch particle stabilized Pickering emulsions on the bioaccessibility of curcumin was fully revealed. There are large differences between the bioaccessibility values of curcumin samples obtained by these two models. Simulated small intestinal lipolysis in the pH-stat model revealed that the bioaccessibility of curcumin encapsulated in the Pickering emulsion was 27.6%, which was larger than 22.1% for free curcumin suspended in the bulk oil phase. The bioaccessibility of curcumin was 50.7% in the emulsion system and 7.8% in the bulk oil when using the TIM-1 model, which simulated the digestion conditions of the entire human GI tract. The digestion mechanism of the milled starch particle stabilized Pickering emulsions in the upper GI tract was well elucidated by the TIM-1 model. The gradual release and improved dissolution profile of the milled starch particle stabilized Pickering emulsions highlighted their potential as delivery systems for lipophilic bioactive compounds.

Metabarcoding is a powerful, increasingly popular tool for biodiversity assessment, but it still suffers from some drawbacks (specimen destruction, separation, and size sorting). In the present study, we tested a non-destructive protocol that excludes any sample sorting, where the ethanol used for sample preserving is filtered and DNA is extracted from the filter for subsequent DNA metabarcoding. When tested on macroinvertebrate mock communities, the method was widely successful but was unable to reliably detect mollusc taxa. Three different protocols (no treatment, shaking, and freezing) were successfully applied to increase DNA release to the fixative. The protocols resulted in similar success in taxa detection (6.8-7 taxa) but differences in read numbers assigned to taxa of interest (33.8%-93.7%). In comparison to conventional bulk sample metabarcoding of environmental samples, taxa with pronounced exoskeleton and small-bodied taxa were especially underrepresented in ethanol samples. For EPT (Ephemeroptera, Plecoptera, Trichoptera) taxa, which are important for determining stream ecological status, the methods detected 46 OTUs in common, with only 4 unique to the ethanol samples and 10 to the bulk samples. These results indicate that fixative-based metabarcoding is a non-destructive, time-saving alternative for biodiversity assessments focussing on taxa used for ecological status determination. However, for a comprehensive assessment on total invertebrate biodiversity, the method may not be sufficient, and conventional bulk sample metabarcoding should be applied.

To understand cellular functionalities, it is essential to unravel spatio-temporal patterns of molecular distributions and interactions within living cells. The technological progress in fluorescence microscopy now allows in principle to measure these patterns with sufficient spatial resolution. However, high resolution imaging comes with long acquisition times and high phototoxicity. Therefore, physiological live cell imaging is often unfeasible and chemical fixation is employed. Yet, fixation methods have not been rigorously investigated, in terms of pattern preservation, at the resolution at which cells can now be imaged. A key parameter for this is the time required until fixation is complete. During this time, cells are under unphysiological conditions and patterns decay. We demonstrate here that formaldehyde fixation takes more than one hour for cytosolic proteins in cultured cells. Other small aldehydes, glyoxal and acrolein, did not perform better. Associated with this, we found a distinct displacement of proteins and lipids, including their loss from cells. Fixations using glutaraldehyde were faster than four minutes and retained most cytoplasmic proteins. Surprisingly, autofluorescence produced by glutaraldehyde was almost completely absent with supplementary addition of formaldehyde without compromising fixation speed. These findings indicate, which cellular processes can actually be reliably imaged after a certain chemical fixation.

Chemical fixation is nearly indispensable in the biological sciences, especially in circumstances where cryo-fixation is not applicable. While universally employed for the preservation of cell organization, chemical fixatives often introduce artifacts that can confound identification of true structures. Since biological research is increasingly probing ever-finer details of the cellular architecture, it is critical to understand the nanoscale transformation of the cellular organization due to fixation both systematically and quantitatively. In this work, we employed Partial Wave Spectroscopic (PWS) Microscopy, a nanoscale sensitive and label-free live cell spectroscopic-imaging technique, to analyze the effects of the fixation process through three commonly used fixation protocols for cells in vitro. In each method investigated, we detected dramatic difference in both nuclear and cytoplasmic nanoarchitecture between live and fixed states. But significantly, despite the alterations in cellular nanoscale organizations after chemical fixation, the population differences in chromatin structure (e.g. induced by a specific chemotherapeutic agent) remains. In conclusion, we demonstrated that the nanoscale cellular arrangement observed in fixed cells was fundamentally divorced from that in live cells, thus the quantitative analysis is only meaningful on the population level. This finding highlights the importance of live cell imaging techniques with nanoscale sensitivity or cryo-fixation in the interrogation of cellular structure, to complement more traditional chemical fixation methods., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Sample preparations for transmission electron microscopy of virulent Mycobacterium tuberculosis are usually performed with chemical fixation using glutaraldehyde (GA) in a biosafety area followed by post-fixation with aqueous osmium tetroxide (OT) in a conventional laboratory outside the biosafety area. Freeze-substitution with osmium-acetone (OA) at ultralow temperature (-85°C) has been shown to provide high quality final images and preserves cellular structures intact. However, some preparation procedures for freeze-substitution often require large fixed devices for freezing in a special laboratory. We have reported a novel freeze-substitution preparation method that can be performed using a portable device in a biosafety cabinet at biosafety level (BSL) 3 areas. Here, as a next step, we examined whether images obtained from rapid freeze-substitution (RFS) after fixation with glutaraldehyde (GA>RFS) are of comparable quality to those obtained using standard RFS. GA>RFS provided excellent preservation of mycobacterial cell ultrastructure, including visualization of cytoplasmic ribosomes, DNA fibers, and the outer membrane. The average number of ribosomes per cubic micrometer counted on RFS and GA>RFS was not significantly different (6987.8±2181.0 and 6888.9±1799.3, respectively). These values were higher, but not significantly so, than those obtained using conventional chemical fixation (5018.7±2511.3). This procedure may be useful for RFS preparation of unculturable mycobacteria strains or virulent strains isolated in laboratories that cannot perform RFS., (© 2013.)

This protocol describes the formaldehyde fixation of Drosophila tissues other than egg chambers. It provides a general approach to fixing tissue or cells onto microscope slides in a manner compatible with whole-mount fluorescent in situ hybridization (FISH) analysis. The procedure includes a simple formaldehyde fixation in buffer, followed by postfixation in cold ethanol. Postfixation of the tissue in methanol or ethanol can markedly improve permeability. In some instances, it may be preferable to fix tissue onto coverslips, because microscope optics are usually designed to optimize imaging immediately adjacent to the coverslip. On the other hand, coverslips are much more fragile and harder to manipulate.

High-pressure freezing (HPF) followed by freeze-substitution is a valuable method for specimen preservation for transmission electron microscopy (TEM) in Drosophila. However, not all projects require this level of precision. In addition, some tissues are too large to fit into the HPF specimen carriers, and some fly tissues such as eyes and ovaries do not freeze well. This protocol describes a trialdehyde fixation procedure for embryos, to be used in situations where optimal preservation is not required or when HPF is not an option. Because the vitelline membrane is impermeable to aqueous solvents, it is necessary to either mechanically disrupt it or render it permeable by treatment with organic solvents. Good ultrastructural preservation has been achieved by puncturing embryos immersed in fixative with extremely sharp tungsten needles, as described here.

This protocol describes paraformaldehyde fixation of Drosophila testes. The procedure preserves F-actin-containing structures because it does not involve the use of methanol. Thus, it is particularly suitable for contractile ring and fusome visualization. However, it does not preserve cell morphology and microtubules as efficiently as methanol-acetone fixation and does not permit clear detection of the Y loops.

Formaldehyde, one of the most toxic organic compounds, is produced and processed in human cells. The level of human endogenous formaldehyde is maintained at a low concentration (0.01-0.08 mmol L(-1) in blood) under physiological conditions, but the concentration increases during ageing (over 65 years old). Clinical trials have shown that urine formaldehyde concentrations are significantly different between elderly Alzheimer's patients (n=91) and normal elderly volunteers (n=38) (P<0.001). Abnormally high levels of intrinsic formaldehyde lead to dysfunction in cognition such as learning decline and memory loss. Excess extracellular and intracellular formaldehyde could induce metabolic response and abnormal modifications of cellular proteins such as hydroxymethylation and hyperphosphorylation, protein misfolding, nuclear translocation and even cell death. This cellular response called formaldehyde stress is dependent upon the concentration of formaldehyde. Chronic impairments of the brain resulted from formaldehyde stress could be one of the mechanisms involved in the process of senile dementia during ageing.

Zinc salt-based fixation (ZBF) has proved advantageous in histochemical analyses conducted on intact tissues but has not been exploited in flow cytometry procedures that focus on quantitative analysis of individual cells. Here, we show that ZBF performs equally well to paraformaldehyde in the preservation of surface epitope labeling and forward and side scatter parameters as measured by flow cytometry. ZBF-fixed mouse epithelial keratinocytes exhibit a staining pattern for the surface markers Sca-1, CD34 and alpha6 integrin that is highly analogous to live cells. Furthermore, ZBF also preserves DNA allowing subsequent quantitative PCR analysis or labeling for incorporation of the thymidine analog EdU following surface and intracellular epitope staining. Finally, ZBF treatment allows for long-term storage of labeled cells with little change in these parameters. Thus, we present a protocol for zinc salt fixation of cells that allows for the simultaneous analysis of DNA and intracellular and cell surface proteins by flow cytometry.

In the collection of field materials to test for the presence of arboviruses, samples must be appropriately maintained to detect arboviral nucleic acids. In austere field conditions this is often difficult to achieve because, during routine specimen processing, storage, and shipping viral RNA degradation could result in detection failure. RNA extraction reagents, while used commonly for their intended purpose of stabilizing RNA during the extraction process, have not been assessed fully for their potential to stabilize RNA before extraction. The potential for virus stabilization at varying temperatures and periods of time remains unknown. Accordingly, the ability of buffer AVL (Qiagen, Valencia, CA), an RNA extraction reagent, to stabilize viral suspensions of dengue, Venezuelan equine encephalitis and Rift Valley fever viruses was evaluated. The ability of buffer AVL to stabilize each viral suspension was examined at 32, 20, 4, and -20 degrees C. RNA in samples placed in buffer AVL was stable for at least 48h at 32 degrees C and refrigerating samples prolonged stabilization. Additionally, placing the sample/buffer AVL mixture at either 4 or -20 degrees C stabilized samples for at least 35 days. When combined with the ability of buffer AVL to inactivate viral samples, this provides the ability to collect and handle potentially infectious samples in a safe way that also provides sample stabilization.

Bovine Type I collagen was investigated, building on a large scale computer model of a collagen fibril in water, and focusing on two stages of the leather manufacturing process. The effects of different salts (NaCl, CaCl(2), and Na(2)SO(4)) on the swelling behavior of collagen at low pH (the pickling process) were studied. The salts suppress the swelling of the fibrils at low pH and we find specific stabilizing influences for CaCl(2) and Na(2)SO(4), due to weak Ca(2+)/Cl(-) and strong SO(4) (2-)/lysine/arginine interactions, respectively. Using state-of-the-art sampling techniques, such as the metadynamics algorithm, to allow an efficient exploration of configuration space, we were able to investigate the effect of polyacrylate and poly(methyl acrylate) - two polymeric retanning agents - on the fibril. Both polymers interact with the ammonium groups on the surface, but polyacrylate shows significantly stronger interactions. We suggest that it is this stronger interaction that contributes to the reduced suitability of PAA as a tanning agent.

Formaldehyde (FA) is a mutagen that, at high concentrations and long durations, has been reported to cause nasal cancer in rats and in some humans. The level of FA-induced modified DNA in nasal cells should serve as a biomarker of FA exposure and effect. In the present study, a high-performance liquid chromatography (HPLC)-ultraviolet (UV) method at 254 nm was developed and optimized to detect and quantify hydroxymethyldeoxynucleosides after the isolated DNA in exposed human nasal epithelial cells (HNEC) was enzymically digested. Normal and modified deoxynucleosides were successfully resolved from one another and from tissue and enzyme blank interferences. The viability of HNEC exposed to FA in solution for 24 h decreased, and there was a linear dose response between % nonviability and FA dose from 10 to 500 microg/mL. Amounts of 18.0 +/- 1.5 pmol N6-dA and 12.0 +/- 1.2 pmol N2-dG derivatives were determined in a 10 microL injection after 1.4 x 10(7) HNEC (106 microg DNA) were exposed to 500 microg/mL in solution. The respective tissue concentrations in pmol hydroxymethyldeoxynucleoside/mg DNA were 170 +/- 14 and 113 +/- 11. The lower quantifiable limits were about 97 and 88 pmol/mg DNA, respectively. Diffusive exposure of HNEC to air FA up to 100 ppm (v/v) for 24 h did not produce quantifiable hydroxymethylnucleosides. FA-modified deoxynucleosides may be useful biomarkers for FA exposure in biological monitoring samples taken by nasal lavage or brush biopsy.

The development of appropriate technologies for the treatment of formaldehyde discharged into the environment is important to minimize its impact. Aerobic systems have been employed, although alternative anaerobic treatments have also been widely studied, mainly due to their low energy consumption and sludge production. However, toxic substances can lead to disturbances in anaerobic reactors. Some research has already been developed on formaldehyde anaerobic biological treatment, but no consensus has yet been reached about its behavior nor has the most efficient system been identified. Aiming at finding supporting evidence for this issue, therefore, this study investigated the degradation and toxicity of formaldehyde in a Horizontal-Flow Anaerobic Immobilized Sludge Reactor. Formaldehyde concentrations of 26.2-1158.6 mg HCHO/L were applied in the reactor, resulting in formaldehyde and chemical oxygen demand removal efficiencies of 99.7% and 92%, respectively. Volatile fatty acids with up to five carbons, found during the degradation of formaldehyde, are believed to indicate that the degradation followed routes unlike those suggested in the literature, which reports the formation of intermediates such as methanol and formic acid. The Monod kinetic model adhered to the experimental data well, with apparent kinetic parameters estimated as r(app)max) = 2.79 x 10(-3) mg HCHO/mg SSVh and K (app)(s) = 242.8 mg HCHO/L.

TWO BACTERIAL STRAINS: Arthrobacter sp. and Sphingomonas paucimobilis were isolated from soil by enrichment cultures using dimethylphthalate (DMP) or monomethylphthalate (MMP) as sole carbon source, respectively. DMP was rapidly transformed by an Arthrobacter sp. culture with formation of MMP and phthalic acid (PA) which is further degraded. This strain was unable to hydrolyse MMP. A mechanism of degradation of DMP was proposed with two ways: DMP-->PA and DMP-->MMP. The S. paucimobilis strain hydrolyses only MMP and a coculture of the two strains allowed a complete degradation of DMP.

The effect and mechanism of inorganic carbon (IC) on the biodegradation of dimethyl phthalate (DMP) by a green microalga Chlorella pyrenoidosa was investigated. It was indicated that DMP could be used as the sole carbon source to support the slow heterotrophic growth of C. pyrenoidosa, but both the growth of C. pyrenoidosa and the biodegradation rate of DMP were obviously increased when initial inorganic carbon concentration (IC) was increased from 0.6 to 23.7 mg/l. Phthalic acid (PA) was found to be an intermediate product of DMP biodegradation and accumulated in the culture solution, which caused a sharp decrease in pH of medium and inhibited both the growth of alga and the biodegradation of DMP. The role of IC for improving the biodegradation of DMP was both to supply a favorite carbon source to support the rapid growth of alga and to mitigate the decrease of pH because of the production of PA. A suggested second-order kinetic equation of organic pollutant biodegradation by microalgae (-dC/dt = KNr) fitted well with the experimental data and the correlation coefficients were all above 0.9. The second-order constant (K) apparently declined with the increase of initial IC because lower ratio between organic carbon from DMP and IC was used to support the growth of alga when initial IC increased.

A Bacillus sp. which is capable of degrading dimethylphthalate (DMP) was immobilized in calcium alginate and polyurethane foam for efficient and long term degradation of DMP. Freely suspended cells (10(12) cfu ml-1) degraded a maximum of 20 mM DMP. Whereas, alginate-(10(12)cfu g-1 beads) and polyurethane foam-entrapped (0.34 x 10(6-9) cfu g-1 foam cubes) cells degraded a maximum of 40 mM DMP within 12-15 days of incubation. Polyurethane foam-entrapped cells degraded 30 mM of DMP at 4 days and alginate-entrapped cells degraded within 10 to 12 days of incubation irrespective of the cell population. When the initial concentration of DMP increased to 50 mM, the DMP degrading ability of the immobilized cells was not increased even after 20 days. Repeated batch cultures by alginate-entrapped cells with initial 35 mM DMP loading could be reused for a maximum of 20 cycles. However, the degradation rate was gradually decreased when the beads were reused for more than 15 cycles. On the other hand, the foam-entrapped cells, with the same initial DMP loading there was no decrease in DMP degrading ability and could be reused for more than 20 cycles. The packed bed reactor with alginate-entrapped cells (1 x 10(10-12) cfu g-1 bead) could be continuously operated for 7-8 days with an initial 25 mM DMP at a flow rate of 50 ml h-1. Whereas, the polyurethane foam-entrapped cells (1 x 10(6-9) cfu g-1 foam cubes) could be operated continuously for more than 90 days with the same initial DMP loading at a flow rate of 100 ml h-1. Thus the enhanced degradation of DMP could be achieved by immobilizing the cells of Bacillus sp. in calcium alginate and polyurethane foam as compared to that of freely suspended cells.

The article is devoted to the medical biological problem that is the impact of endogenic aldehyde (methylenglucol) on tissue structures with the purpose of defending them from ischemic damage by reverse reduction of intensity of metabolic reactions, which is conditioned by reverse inhibition of the oxidation processes phosphorylation connected with reduction of losses and adenosine 5-triphosphate forming. It is ascertained that until methylenglucol in the ischemic tissue of concrete organ hasn't reduced to a certain critical level there is still an opportunity to reanimate this organ.

In histological studies using retinas, eyes are commonly fixed with aldehyde derivatives administered by immersion or perfusion. However, the histology of rat retinas chemically fixed as a whole eye is typically inferior to the histology of retinas that are immediately fixed after acute dissection from the rest of the eye. Chemical fixation without dissection often results in neuronal swelling resembling excitotoxic damage induced by ischemia because the retina is protected by the sclera and is thus poorly accessible to immersion or perfusion fixation techniques. In order for the acute dissection technique to work properly, it must be completed in a timely manner, which may be difficult under some circumstances. Microwave irradiation is an alternative method for fixing tissues that are inaccessable to chemicals. We examined the effectiveness of microwave irradiation of the whole eye as a substitute for acute retinal dissection. To study the feasibility of microwave methods, we compared retinal morphology using microwave irradiation to morphology using conventional immersion fixation methods. Eyes were removed from rats, placed in a container with 2 or 20 ml artificial cerebrospinal fluid (aCSF) and irradiated with a household microwave oven. For morphological comparison, control eyes were immersed in a chemical fixative containing 1% paraformaldehyde and 1.5% glutaraldehyde. All eyes were embedded in araldite for evaluation by light microscopy. Retinal segments acutely isolated before immersion fixation revealed intact histology whereas retinal segments exposed to 60 min of simulated ischemia showed severe neuronal degeneration. Using an immersion technique, the retinas of chemically fixed whole eyes showed neuronal swelling similar to excitotoxic ischemic damage, suggesting that conventional immersion methods provide poor whole eye fixation. The neuronal degeneration observed with conventional immersion fixation was not found in retinas of whole eyes fixed with 20 sec of microwave irradiation. During microwave irradiation the temperature in the bathing aCSF rose to 55-72 degrees C. In some eyes, overcooking produced chromatin clumping and a small loss of contrast in staining. Although nuclear clumping and diminished staining occasionally result from overcooking, ischemic damage is well controlled with microwave fixation of enucleated eyes. When the optimal conditions are defined, microwave fixation may be preferable for retinal histology if chemical fixation following acute dissection is not feasible., (Copyright 2000 Academic Press.)

Most model experiments concerning tissue fixation have used low concentrations of fixatives and proteins. Here, high concentrations (up to 32%) of bovine serum albumin (BSA) were reacted with formaldehyde (1-20%) and glutaraldehyde (0.5-4%). Gels were formed between 16% BSA and 10-20% formaldehyde at room temperature, but not with percentages of formaldehyde lower than 4%. Microwave application or heating in a water bath to 50 degrees C gave a gel from 1 to 20% formaldehyde. Sixteen percent BSA also gave a gel with glutaraldehyde from 0.5 to 4%. Cone and plate viscometry showed rapidly increasing viscosity at 4% formaldehyde and 16% BSA at room temperature. At 50 degrees C, gels formed at a low concentration of formaldehyde. Tissue fixation in which the local concentrations of protein may be in excess of 30% is probably more complete than in vitro experiments in which low concentrations of reagents have been used to permit subsequent spectrometry. This was confirmed by polyacrylamide gel electrophoresis of liver., (Copyright 1998 Academic Press.)

A study of dimethyl hydrogen phosphite (DMHP) by the National Toxicology Program (NTP) indicated that chronic administration by oral gavage resulted in an increased incidence of neoplastic lesions in the lungs and forestomachs of Fischer 344 rats but not in B6C3F1 mice. The current study was designed to evaluate the metabolic basis, if any, of this species selectivity by studying the metabolism and disposition of [14C]DMHP in the respective strains of rats and mice. Results of this study indicate that DMHP administered at a range of dose of 10-200 mg/kg was readily and near completely absorbed from the gastrointestinal tracts of rats and mice. DMHP-derived radioactivity was eliminated primarily as CO2 in the expired air, 44-57%, and urine, 28-49%, and very little was collected in feces, 1-2%, or as volatile organics, 2-3%. DMHP-derived radioactivity was widely distributed in tissues of rats and mice, with the highest concentrations observed in the liver, kidneys, spleen, lungs, and forestomach, and the lowest in brain, skeletal muscle, and adipose tissue. The disappearance of radioactivity from mouse tissues was approximately twice as rapid as from rat tissues. In vitro, DMHP was metabolized to formaldehyde by the microsomal fractions of liver, lungs, kidneys, forestomach, and glandular stomach. In vivo, DMHP was metabolized to the product of demethylation, monomethyl hydrogen phosphite (MMHP), which was excreted in urine. Results of this study indicate that the NTP carcinogenicity study with DMHP was carried out within the dose range in which the absorption, metabolism, and disposition of DMHP are linear in both species. Apparent species-dependent differences in the metabolism and disposition of DMHP are limited to the more rapid metabolism and elimination by the mouse. Therefore, the species-dependent variations in the carcinogenicity of DMHP are most likely attributable to factors other than metabolism and disposition.

The clinical results of biological vascular grafts have been unsatisfactory. The poor results of these vascular grafts may be attributed to the fixatives, aldehydes, used in fixing tissues. In an attempt to overcome this problem, a new fixative, epoxy compound, has recently been used to fix biological vascular grafts. The study was undertaken to investigate the crosslinking characteristics, fixation index and denaturation temperature, of various porcine arteries fixed with an epoxy compound. The porcine arteries investigated in the study were the common carotid artery, internal thoracic artery, abdominal aorta, and saphenous artery. In addition, the effects of sonication on the porcine arteries before fixation on their crosslinking characteristics were analyzed. The fresh and glutaraldehyde-fixed arteries were used as controls. It was noted that glycine, proline, and alanine were the most abundant amino acids found in the porcine internal thoracic artery. In the amino acid analysis, it was observed that the amino acids in the porcine arteries reacted with epoxy compound or glutaraldehyde were lysine, hydroxylysine, histidine, and arginine. Of these amino acids, lysine was the most reactive. In general, the fixed arteries were relatively stiffer than their fresh counterparts. The fixation indices and denaturation temperatures of various porcine arteries were comparable throughout the entire fixation process. The amounts of free amino groups of the sonicated arteries were significantly lower than those of their unsonicated counterparts (p < 0.05). It is speculated that the diminishing free amino groups of the sonicated arteries may be attributed to the removal of the destroyed cell debris and adherent proteins of the arteries after sonication. However, it was learned that sonication on the porcine arteries before fixation did not seem to affect their fixation indices and denaturation temperatures. The results obtained in this study may help one in selecting the raw materials for developing a small-diameter biological vascular graft

The use of glutaraldehyde as a fixative in bioprostheses and drug delivery matrices is reviewed. The chemistry of glutaraldehyde cross-linking and its effect on the biological performance of a number of bioprostheses such as tissue heart valves, vascular grafts, pericardial patches, tendon grafts and drug delivery matrices are examined.

The immunohistochemical staining of vimentin in paraffin-embedded sections from adult cattle, rabbit, rat and mouse brain fixed in different fixatives (formaldehyde, methacarn, ethanol) was examined using two monoclonal antibodies and a polyclonal antiserum. In non-trypsinized formaldehyde-fixed tissue sections both monoclonal antibodies and the polyclonal antibodies failed to stain vimentin. Following trypsinization of formaldehyde-fixed sections of the four species the meninges, endothelial cells of blood vessels, ependymal cells and the stroma of the choroid plexus were labelled by the monoclonal and polyclonal antibodies used. Astrocytes and Bergmann glial fibers in pretrypsinized formaldehyde-fixed sections from cattle, rabbit and rat brain, however, showed only weak staining. Fixation of cattle and rat brain in methacarn markedly improved the vimentin immunoreactivity of astrocytes and Bergmann glial fibers. The best fixative for the preservation of immunoreactive determinants of vimentin in astrocytes and Bergmann glial fibers in cattle, rabbit and rat brain was ethanol. In brain tissue from mice both monoclonal antibodies labelled only mesoderm-derived tissue components, but did not recognize vimentin in astrocytes and Bergmann glial fibers. Pre-heating formaldehyde-fixed sections from cattle, rabbit and rat brain in a microwave oven prior to the immunohistochemical reaction resulted in an enormous enhancement of vimentin staining of mesoderm-derived tissues, of astrocytes and bergmann cell fibers.

The diffusion of fixatives is slow. Early work using plasma gels and animal tissues showed the distance penetrated by a fixative to be a simple function of the fixation time but this relation has not been established in human tissues. The rates of diffusion into whole human spleens were measured for three primary fixatives over periods ranging from one to 25 days. A positive correlation was demonstrated between penetration distance (mm) and fixation time (hours). The diffusion rates were slower than those in previous studies. These results have possible implications for the handling of surgical specimens.