Effect of fixation on immunolocalization of transferrin and albumin in the liver of the adult rat

The influence of the fixation procedure on the localization of albumin and transferrin in adult rat liver has been carried out using an indirect immunoperoxidase technique at the light and electron microscopic levels. Perfusion and immersion fixations with different concentrations of paraformaldehyde (with or without addition of glutaraldehyde) have been investigated. According to the mode of fixation (perfusion versus immersion) and the concentration of the fixative, the number of albumin and transferrin containing hepatocytes could vary from 10% to 100%, and different labeling patterns could be observed at the electron microscopic level. For the same concentration of fixative, a perfusion fixation induces a less intense labeling than an immersion fixation. Thus similar results are obtained after immersion fixation in 6% paraformaldehyde + 0.25% glutaraldehyde or after perfusion fixation in 4% paraformaldehyde + 0.025% glutaraldehyde. Similar data are noticed after immersion fixation in 4% paraformaldehyde or after perfusion fixation in 1% paraformaldehyde + 0.025% glutaraldehyde. Moreover, perfusion fixation induced a more fine cell structure preservation than immersion fixations and avoided the appearance of zones of fixation.     

Fixation of vervet monkey oral mucosa for ultrastructural investigation--TEM

This study was undertaken to determine optimal fixation procedure for vervet monkey (Cercopithecus pygerythrus) oral mucosa. Perfusion and immersion fixation were investigated using glutaraldehyde and glutaraldehyde-paraformaldehyde fixatives with either a phosphate or sodium cacodylate buffer as vehicle and with osmolarities varying from 2010 to 320 mosm. Good fixation could not be obtained uniformly or consistently by perfusion. Vervet monkey oral mucosa is best fixed by first perfusing the head and neck of the animal with 250-500 ml 0.9% saline containing Procaine-HCl and heparin, followed by decapitation and immersion of the head in a 2.5% glutaraldehyde: 2% paraformaldehyde: 0.02 M sodium cacodylate buffered fixative (900 mosm) at 4 C for 24 hr.     

Fixation of Vervet Monkey Oral Mucosa for Ultrastructural Investigation-Tem

This study was undertaken to determine optimal fixation procedure for vervet monkey (Cercopithecui pygerythrta) oral mucosa. Perfusion and immersion fixation were investigated using glutaraldehyde and glutaraldehyde-paraformaldehyde fixatives with either a phosphate or sodium cacodylate buffer as vehicle and with osmolalities varying from 2010 to 320 mosm. Good fixation could not be obtained uniformly or consistently by perfusion. Vervet monkey oral mucosa is best fixed by first perfusing the head and neck of the animal with 250-500 ml 0.9% saline containing Procaine-HCl and heparin, followed by decapitation and immersion of the head in a 2.5% glutaraldehyde: 2% paraformaldehyde: 0.02 M sodium cacodylate buffered fixative (900 mosm) at 4 C for 24 hr.     

Fixation of Vervet Monkey Oral Mucosa for Ultrastructural Investigation–Tem

This study was undertaken to determine optimal fixation procedure for vervet monkey (Cercopithecui pygerythrta) oral mucosa. Perfusion and immersion fixation were investigated using glutaraldehyde and glutaraldehyde-paraformaldehyde fixatives with either a phosphate or sodium cacodylate buffer as vehicle and with osmolalities varying from 2010 to 320 mosm. Good fixation could not be obtained uniformly or consistently by perfusion. Vervet monkey oral mucosa is best fixed by first perfusing the head and neck of the animal with 250-500 ml 0.9% saline containing Procaine-HCl and heparin, followed by decapitation and immersion of the head in a 2.5% glutaraldehyde: 2% paraformaldehyde: 0.02 M sodium cacodylate buffered fixative (900 mosm) at 4 C for 24 hr.     

Immersion and perfusion fixed rat adrenal medulla: The problem of mixed cells,clear cells,and the mode of secretion

Summary The adrenal medulla of the rat was studied utilizing various methods of fixation. In adrenal medulla specimens after immersion fixation either with glutaraldehyde or osmium tetroxide, elements such as mixed, clear, syncytial, or plasmodial cells, believed to be of artifactual origin, are observed in all of this material examined. These elements are absent in the specimens prepared by perfusion fixation. In specimens prepared by immersion fixation, secretory granules are found in close proximity to the plasma membrane; this localization is infrequent after perfusion fixation.Current theories of the mechanism of secretion of adrenal medullary hormones are discussed on the basis of our results. This investigation demonstrates the advantage and necessity of perfusion fixation in the study of the adrenal medulla.Supported by the Deutsche Forschungsgemeinschaft, grant No. Fo 77/1.     

Quantitative assessment of cellular changes provoked by microwave enhanced fixation of parathyroids

Rat parathyroids fixed by microwave enhancement, i.e. microwave irradiation in the presence of glutaraldehyde for 8 s and postfixation with OsO4 after a delay of 5 min, were compared with parathyroids fixed by perfusion with glutaraldehyde followed by immersion in glutaraldehyde and finally in OsO4. Morphometric analysis revealed that microwave enhanced fixation led to a larger mean cell volume, to larger cell surface area, and to larger surface area in membranes of RER and secretory granules. Though it is not known by which method parathyroid cells are conserved closer to the living state it is obvious that microwave enhanced fixation retains more membranes but provokes centrifugal dislocation of membranes mimicking exocytosis.     

Quantitative assessment of cellular changes provoked by microwave enhanced fixation of parathyroids

Summary Rat parathyroids fixed by microwave enhancement, i.e. microwave irradiation in the presence of glutaraldehyde for 8 s and postfixation with OsO₄ after a delay of 5 min, were compared with parathyroids fixed by perfusion with glutaraldehyde followed by immersion in glutaraldehyde and finally in OsO₄. Morphometric analysis revealed that microwave enhanced fixation led to larger mean cell volume, to larger cell surface area, and to larger suface area in membranes of RER and secretory granules. Though it is not known by which method parathyroid cells are conserved closer to the living state it is obvious that microwave enhanced fixation retains more membranes but provokes centrifugal dislocation of membranes mimiking exocytosis.     

Hydrolysis of rice hull by crosslinked Aspergillus niger cellulase

A. niger cellulase was crosslinked by glutaraldehyde to obtain a heat-stable enzyme preparation for rice hull cellulose hydrolysis. Under optimized crosslinking conditions of 0.12 M glutaraldehyde, pH 7.0, temperature 40 degrees C and at 45 min of crosslinking, a preparation having 15% more activity than free enzyme was obtained which also had considerable improvement in heat stability at 65 degrees C and 70 degrees C. Whereas the free enzyme lost 80% of its activity in 4 h at 65 degrees C, the crosslinked preparation lost only 30% activity. The crosslinked preparation hydrolyzed cellulosic biomass more effectively giving 2.2 mg/ml glucose and 52% corresponding saccharification in 4 h at 65 degrees C as compared to 14% saccharification by free enzyme under similar conditions.     

Parathyroid cell variants may be provoked during immersion fixation

Parathyroid glands of cattle, dogs, cats, mice and rats were immersed in glutaraldehyde or mixtures consisting of glutaraldehyde, formaldehyde and acrolein in either Na-phosphate, Na/K-phosphate or Na-cacodylate buffer, and postfixed with OsO4 in the same buffers or, alternatively, in s-collidine. Excellent preservation of bovine, feline and murine parathyroid glands was achieved with fixation mixtures containing 1% glutaraldehyde, 1.5-2% formaldehyde and 2.5-5% acrolein in 0.1 M Na-cacodylate with or without Ca2+ and Mg2+, Na-phosphate or Na/K-phosphate at 4 degrees C followed by postfixation with 1% OsO4 in the same buffers or in s-collidine containing sucrose, Ca2+ and Mg2+. This procedure largely abolished the occurrence of parathyroid cell variants. Bovine parathyroid glands were also satisfactorily preserved with 1% glutaraldehyde and 2% formaldehyde whereas 1% glutaraldehyde and 2.5 or 5% acrolein, lower or higher buffer osmolarity, or immersion at room temperature led to vacuolization of RER and to breakdown of membranes. In contrast, all fixation protocols led to the formation of dark and light cell variants and to multinucleated syncytial cells in dog and rat parathyroids. The results thus show that parathyroid cell variants arise during immersion fixation and that aldehydes, buffers and temperature are important factors for provoking parathyroid cell variants.     

Glycosyltransferases in plant and animal tissues effects of fixation and lead on enzyme activity.

As the initial step toward the cytochemical localization of glycosyl-transferases in situ, biochemical determinations of these enzyme activities from onion root tips and L1210 cells were performed before and after fixation as well as in the presence of lead ions. Glycosyltransferase activity from roots fixed in buffered formaldehyde or glutaraldehyde before homogenization decreased as the concentration of the fixative or fixation time was increased. Formaldehyde fixation was less inhibitory than glutaraldehyde; 35% of the glycosyltransferase activity was retained after 30 min fixation in 2% formaldehyde while 25% of the enzyme activity remained after a similar fixation in glutaraldehyde. Substantially higher levels of L1210 cell glycosyltransferase activity were retained after a 30 min 2% formaldehyde fixation (60% sialyltransferase; 82% galactosyltransferase), but inhibition by glutaraldehyde was similar to that observed for onion root galactosyltransferase. Glycosyltransferase from formaldehyde-fixed roots was inhbited 35% by lead nitrate, but sialytransferase from formaldehyde-fixed L1210 cells was unaffected by lead ions. These findings are encouraging for further studies aimed at the development of cytochemical technique to localize glycosyltransferase in plant and animal tissues.     

Cytochemical demonstration of adenylate cyclase in cardiac muscle: effect of dimethyl sulfoxide.

Adenylate cyclase (AC) activity was evaluated after perfusion fixation of rat and dog myocardium with 4% paraformaldehyde (PFA), 2% glutaraldehyde (GA) or a combination of both, in cacodylate buffer. Dimethyl sulfoxide (DMSO) was added to the fixatives and its effect on the preservation of cell organelles and enzyme activity was determined. Adenylate cyclase activity was preserved best after fixation with 4% paraformaldehyde but this fixative did not provide for optimal maintenance of structure. Prefixation with 2% glutaraldehyde and 5% dimethyl sulfoxide provided the most effective preservation of both structural and enzymatic integrity. Precipitation of lead diphosphoimide was the morphologic indicator of sites of adenylate cyclase activity. The most intense precipitate was in the lumen of junctional sarcoplasmic reticulum in close contact with T-tubules and in subsarcolemmal cisternae. Evidence of activity was also seen on the intracellular aspect of the sarcolemmal membrane and in the nexus segment of the intercalated discs. Alloxan was effective as an inhibitor of adenylate cyclase activity only if the concentration of the activating substance sodium fluoride (NaF) was 20 mM or lower.     

Chemical inactivation of HIV on surfaces.

To assess whether alcohol and glutaraldehyde are effective disinfectants against dried HIV the virucidal effects of 70% alcohol (ethanol and industrial methylated spirit) and 1% and 2% alkaline glutaraldehyde were tested against cell associated and cell free HIV dried on to a surface. Virus stock (100 microliters) or 10,000 cultured C8166 T lymphocytes infected with HIV were dried onto sterile coverslips and immersed in 2% and 1% alkaline glutaraldehyde and 70% ethanol for 30 seconds and one, two, four, and 10 minutes, there being an additional time point of 20 minutes for cell free virus disinfected with 70% industrial methylated spirit. In addition, virus stock in neat serum was tested with 1% and 2% alkaline glutaraldehyde to see whether the fixative properties of glutaraldehyde impair its virucidal properties. Virus activity after disinfection was tested by incubating the coverslips (cell associated virus) or the coverslips and sonicated cell free virus with C8166 T lymphocytes. The lymphocytes were examined for the formation of syncytia and HIV antigens were assayed in the culture fluid. Both 2% and 1% alkaline glutaraldehyde inactivated cell free HIV within one minute; 2% alkaline glutaraldehyde also inactivated cell free virus in serum within two minutes, but a 1% solution was ineffective after 15 minutes'' immersion. Cell associated HIV was inactivated by 2% alkaline glutaraldehyde within two minutes. Seventy per cent industrial methylated spirit failed to inactivate cell free and cell associated HIV within 20 and 15 minutes, respectively, and 70% ethanol did not inactivate cell free virus within 10 minutes. Seventy per cent industrial methylated spirit and ethanol are not suitable for surface disinfection of HIV. Fresh 2% solutions of alkaline glutaraldehyde are effective, but care should be taken that they are not too dilute or have not become stale when used for disinfecting HIV associated with organic matter.     

Demonstration of Pulmonary Surfactant by Tracheal Injection of Tricomplex Salt Mixture: Electron Microscopy

Laboratory animals were perfused with glutaraldehyde through the right ventricle before filling the whole lung intratracheally with a solution of 0.05 N Pb(NO₃)₂ and K₃Fe(CN)₆ and incubating it at 37 C for 30 min. An electron-dense reaction product on the surface of epithelial cells and in certain well-localized regions of the alveolar septa results. Nonspecific, intracellular, precipitates do not occur. Lungs which are sliced before glutaraldehyde fixation and immersed in the tricomplex salt mixture show patchy localization of reaction product. Diffuse, electron-dense deposits measuring 100-2000 mμ in diameter are seen in the cytoplasm of epithelial, endothelial and interstitial cells although occasionally there is localization on the surface of the epithelial cells and in specific sites within cells. Injection of the whole lung with tricomplex salt mixture after fixation with glutaraldehyde by vascular perfusion is a better method for the ultrastructural demonstration of pulmonary surfactant than immersion fixation and staining.     

Improved histological localization of GABA-transaminase activity in rat cerebellar cortex after aldehyde fixation

Summary A method for the chemical fixation of the enzyme GABA-transaminase in nervous tissue is described. It is shown that after perfusion with a formaldehyde/glutaraldehyde fixative, activity of the enzyme in cerebellar cortex is demonstrable whilst cellular morphology is preserved. Results from the improved technique have shown new sites of GABA-transaminase activity in cerebellar cortex. In view of these results a special function for glial cells in this area of brain has been suggested.     

Improved histological localization of GABA-transaminase activity in rat cerebellar cortex after aldehyde fixation.

A method for the chemical fixation of the enzyme GABA-transaminase in nervous tissue is described. It is shown that after perfusion with a formaldehyde/glutaraldehyde fixative, activity of the enzyme in cerebellar cortex is demonstrable whilst cellular morphology is preserved. Results from the improved technique have shown new sites of GABA-transaminase activity in cerebellar cortex. In view of these results a special function for glial cells in this area of brain has been suggested.     

Characterization of dextransucrase immobilized on calcium alginate beads from Leuconostoc mesenteroides PCSIR-4

Immobilization of dextransucrase from Leuconostoc mesenteroides PCSIR-4 on alginate is optimized for application in the production of dextran from sucrose. Dextransucrase was partially purified by ethanol upto 2.5 fold. Properties of dextransucrase were less affected by immobilization on alginate beads from soluble enzyme. Highest activities of both soluble and immobilized dextransucrase found to be at 35 degrees C and optimum pH for activity remain 5.00. Substrate maxima for immobilized enzyme changed from 125 mg/ml to 200 mg/ml. Incubation time for enzyme-substrate reaction for maximum enzyme activity was increased from 15 minutes to 60 minutes in case of immobilized enzyme. Maximum stability of immobilized dextransucrase was achieved at 25 degrees C with respect to time.     

Recovery of maximal isometric grip strength following cold immersion

The purpose of this study was to investigate the effects of various cold immersion durations on maximal grip strength and the subsequent recovery of grip strength. Sixteen healthy men between 20 and 42 years of age participated in this study. Maximal isometric grip strength was measured before, immediately after, and 5, 10, and 15 minutes after cold immersion. Subjects submerged their dominant elbow, forearm, and hand in a cold water whirlpool at 10 degrees C for 5, 10, 15, or 20 minutes. There was a significant decrease in isometric grip strength when the forearm was immersed in 10 degrees C water for durations between 5 and 20 minutes and no recovery of this strength loss for a period of 15 minutes following removal from the cold immersion (p = 0.0001). These findings suggest that clinicians should be aware of the alterations in isometric muscle strength that result from utilizing the temperature and time frames of cold application used in this study.     

Factors influencing the activity of succinate dehydrogenase in membrane preparations from Micrococcus lysodeikticus

1. Some properties of succinate dehydrogenase [succinate-(acceptor) oxidoreductase, EC 1.3.99.1] in membrane preparations from Micrococcus lysodeikticus (N.C.T.C. 2665) were investigated. 2. In the spectrophotometric assay system adopted the reaction velocity was shown to be proportional to the amount of membrane added. Dichlorophenol-indophenol, reduced photochemically in the presence of phenazine methosulphate, or enzymically by the membrane-bound enzyme, was shown to undergo reoxidation in the dark. 3. The membrane-bound enzyme was found to be inactivated at temperatures above 10 degrees C. 4. The specific activity of membrane-bound succinate dehydrogenase was found to increase between two- and three-fold in diluted membrane preparations equilibrated at 0 degrees C for 6h. Membranes treated with sodium deoxycholate showed no enzyme activation on dilution but displayed maximal activity, all activity being sedimentable at 103000g. The increase in specific activity observed on dilution could be partially inhibited by fixation with glutaraldehyde, or by the presence of bovine serum albumin. 5. The addition of Mg(2+) or Ca(2+) ions to membrane suspensions caused an overall depression of enzyme activity. 6. The results suggest the presence of an ;inhibitor' that affects the expression of membrane bound succinate dehydrogenase activity.     

Immobilization of lipase on chitin and its use in nonconventional biocatalysis

Chitin was functionalized with hexamethylenediamine followed by glutaraldehyde activation, and its capacity to bind Candida rugosa lipase was investigated. The loading of 250 units g(-1) support showed to be effective, resulting in a uniform enzyme fixation with high catalytic activity. Both free and immobilized lipases were characterized by determining the activity profile as a function of pH, temperature, and thermal stability. For the immobilized lipase, the influence of the reaction temperature and substrate polarity in nonconventional biocatalysis was also analyzed. Production of butyl esters was found to be dependent on the substrate partition coefficient, which accounts the greatest value for the system butanol and butyric acid. The highest enzyme activity was found for the system butanol and caprylic acid at a reaction temperature of 40 degrees C. Under such conditions, the operational stability tests indicated that a small enzyme deactivation occurs after 12 batches, revealing a biocatalyst half-life of 426.7 h.