Use of isoelectric focusing to determine the isoeletric point of bovine serum albumin after treatment with various common fixatives

Synopsis Isoelectric focusing was used to determine the effects of fixation on the isoelectric point of bovine serum albumin. Aqueous solutions of formaldehyde, -hydroxyadipaldehyde, osmium tetroxide and potassium dichromate produced no change or a relatively small decrease. Glutaraldehyde produced two fractions of fixed protein, the larger with a considerably lowered isoelectric point. The results are interpreted as suggesting that glutaraldehyde reacts rapidly and largely irreversibly, whereas the other fixatives react more slowly and reversibly with bovine serum albumin.     

A comparison of the crosslinking abilities of glutaraldehyde,formaldehyde and α-hydroxyadipaldehyde with bovine serum albumin and casein

Summary The crosslinking effects of formaldehyde, -hydroxyadipaldehyde and glutaraldehyde have been compared by various techniques. Using a micro-Ouchterlony technique with an aldehyde treated bovine serum albumin-rabbit anti-bovine serum albumin system it was found that glutaraldehyde prevented precipitin line formation except at very high titres of antibody. The effects of formaldehyde and -hydroxyadipaldehyde were less marked. Cellulose acetate electrophoresis of aldehyde treated bovine serum albumin showed an increase in mobility compared with the untreated protein. Starch gel electrophoresis of aldehyde fixed liver slices showed no protein loss after glutaraldehyde fixation whereas the other aldehydes permitted proteins to be extracted. Polyacrylamide gel electrophoresis of the aldehyde treated bovine serum albumin showed a little change in mobility after formaldehyde and -hydroxyadipaldehyde treatment and a little polymer formation. Glutaraldehyde on the other hand produced much polymer. These findings were confirmed by gel filtration with Sephadex G-200. Intermolecular crosslinking with glutaraldehyde was dependant on the aldehyde concentration.     

Fixation of proteins by osmium tetroxide potassium dichromate and potassium permanganate

Summary The crosslinking abilities of osmium tetroxide, potassium dichromate and potassium permanganate towards bovine serum albumin and bovine -globulin were investigated by chromatography with Sephadex G-200. Osmium tetroxide had a moderate crosslinking ability towards these proteins, the others had little or none. Chromatography with Sephadex G-50 permitted the oxidative cleavage of the proteins by these oxidative fixation agents to be studied. Potassium permanganate caused much fragmentation of the proteins and destruction of the tyrosine and tryptophan residues. Osmium tetroxide and potassium dichromate caused only a small amount of protein cleavage. These results were corroborated by polyacrylamide gel electrophoresis and viscosimetric studies. The significance of the results for tissue fixation is discussed.     

The effects of mercaptoethanol-formaldehyde on tissue fixation and protein retention

Summary The study compared the effects of mercaptoethanol-formaldehyde and formaldehyde alone, on tissue fixation and protein retention in human and mouse tissues. Shrinkage of tissues and the penetration rate of the fixatives were assessed. The cross-linking ability of the fixatives was determined by viscometry, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and spectrophotometry, using bovine serum albumin and human haemoglobin. Tissues fixed in buffered 0.0025% mercaptoethanol-4% formaldehyde showed good nuclear and cytoplasmic detail, better than those fixed in buffered 4% formaldehyde. There was no significant difference in shrinkage. A mixture of 0.0025% mercaptoethanol-4% formaldehyde penetrated faster into adult liver than 4% formaldehyde. The mean penetration rate (±SE) or coefficient of diffusibility of 0.0025% mercaptoethanol-4% formaldehyde into adult liver was 1.32±0.01 and that of 4% formaldehyde was 1.12±0.06 (p<0.04). Both fixatives diffused more rapidly into mouse liver than into human liver. The cross-linking ability of mercaptoethanol-formaldehyde depends on the concentration of the fixative and the time of fixation. Bovine serum albumin (15%) and 0.1% mercaptoethanol alone formed a gel, whilst electrophoresis showed monomers in the supernatant. Mercaptoethanol (0.1%) also rapidly decreased the absorption at 420 nm, suggesting denaturation. It seems that mercaptoethanol increases the number of thiol groups available to form cross-links with formaldehyde. This study demonstrated that mercaptoethanol-formaldehyde fixed and cross-linked tissues better than formaldehyde at 3 h and 4 h, but not at 1 h and 2 h. The most effective concentration of mercaptoethanol for tissue fixation in 4% formaldehyde is 0.0025%.     

Fluorogenic detection of primary amines in plant histochemistry with fluorescamine: A comparative study on the effects of coagulant and non-coagulant fixatives

Summary The new highly sensitive method of fluorescamine reaction for the topochemical detection of primary amino groups was studied as a substitude of ninhydrin-Schiff's reaction for the localisation of total proteins in plant tissues. The influence of various coagulant and non-coagulatn fixatives on the induction of fluorescamine fluorescence was examined: ethanol, formaldehyde gas and solution, glutaraldehyde, acrolein, osmium tetroxide, Bouin, Rossman, Clarke and Zenker's fluids and FMA were employed. It was found that the use of the fluorogenic method is conditioned by the fixative ability to keep the amino groups disposable and by its capability to reduce the natural autofluorescence of plant material. A detailed account of the fixation methodology demonstrated that non-coagulant acrolein and coagulant mercuric chloride are the most promising fixatives for the use of the fluorescamine reaction in plant histochemistry.     

Fluorogenic detection of primary amines in plant histochemistry with fluorescamine: a comparative study on the effects of coagulant and non-coagulant fixatives.

The new highly sensitive method of fluorescamine reaction for the topochemical detection of primary amino groups was studied as a substitude of ninhydrin-Schiff's reaction for the localisation of total proteins in plant tissues. The influence of various coagulant and non-coagulant fixatives on the induction of fluorescamine fluorescence was examined: ethanol, formaldehyde gas and solution, glutaraldehyde, acrolein, osmium tetroxide, Bouin, Rossman, Clarke and Zenker's fluids and FMA were employed. It was found that the use of the fluorogenic method is conditioned by the fixative ability to keep the amino groups disposable and by its capability to reduce the natural autofluorescence of plant material. A detailed account of the fixation methodology demonstrated that non-coagulant acrolein and coagulant mercuric chloride are the most promising fixatives for the use of the fluorescamine reaction in plant histochemistry.     

The effects of various fixatives on the relative thickness of cellular membranes in the ventral lobe of the rat prostate

Synopsis A densitometric method was utilized in the measurement of the relative thickness of the cellular membranes in the ventral lobe of the rat prostate. Potassium permanganate, glutaraldehyde, osmium tetroxide, and ruthenium tetroxide solutions were used as fixatives. During preparation for electron microscopy, the tissues were given standardized treatments to reduce methodological errors; latex particles were applied to the thin sections to serve as reference particles of a known size. The most remarkable observation of the study was that the densitometric method yielded reproducible results and that the different fixatives gave significantly different values for the relative thickness of cellular membranes. Glutaraldehyde, or glutaraldehyde followed by ruthenium tetroxide post-fixation, gave the highest values for membrane thickness while osmium tetroxide and potassium permanganate gave the lowest values. Glutaraldehyde treatment, prior to osmium tetroxide or potassium permanganate post-fixations, rendered the membranes thicker than after osmium tetroxide and potassium permanganate treatments alone. Ruthenium tetroxide appeared to be very suitable for fixation of cellular membranes.     

PLANT MICROTECHNIQUE: SOME PRINCIPLES AND NEW METHODS

Some easily seen structural features of living plant cells are destroyed or badly distorted by most of the common fixatives and embedding media used in plant histology. In stained sections of plant tissues fixed in FAA (formalin-acetic acid-alcohol mixtures) and embedded in paraffin wax, for example, mitochondria and fine transvacuolar strands of cytoplasm are usually not visible. Many structural features such as these can be preserved, however, with suitable fixatives and embedding media. Specifically we recommend fixation in non-coagulant fixatives (e.g., osmium tetroxide, acrolein, glutaraldehyde, formaldehyde) and the use of plastics as embedding media, and we describe in detail a method of fixation in acrolein and embedding in glycol methacrylate polymer. In a wide range of plant specimens prepared in this way, stained sections 1–3 microns thick showed excellent preservation of tissue and cell structures.     

The effect of chemical fixatives on cell walls of Bacillus subtilis.

Cell walls of Bacillus subtilis were treated with several chemical fixatives which are commonly used preparatory to electron microscopy; i.e., osmium tetroxide, formaldehyde, acrolein, crotonaldehyde, and glutaraldehyde. Dimensional analysis was performed on thin sections of fixed walls from plastic embeddings and, by means of the statistical technique of multiple comparisons, significant differences were found between wall thicknesses from the various fixations. These differences varied with the fixation time and the type of fixative used in the reaction. When compared to embedded walls which had been stained before fixation, the overall effect was a reduction in wall thickness which was attributed to fixative action and not to the embedding or staining processes. The reduction of wall thickness was even more apparent when dimensions of fixed walls were compared to published dimensions of both frozen sections and freeze-etch profiles. Since these fixatives bind to reactive sites within the wall fabric, a change in electrochemical charge density is effected which can be monitored in terms of heavy-metal-binding capacity. Most monoaldehyde fixatives and osmium tetroxide render the wall as reactive, or less reactive, to uranyl acetate as unfixed walls, whereas glutaraldehyde can significantly increase the binding capacity.     

Optimization of gold immunolabeling techniques on ultrathin slides obtained from samples fixed with glutaraldehyde and osmium tetroxide and enclosed in epoxy resin]

An immunogold labeling technique was carried out on plants infected with CMV, fixed with glutaraldehyde and osmium tetroxide and embedded in araldite CY 212. The effect of the type of support-film used, the resin and manipulations of the grids during immunogold steps, were studied and are discussed. The antigenic activity of virus was restored by treating the sections with sodium metaperiodate. The very high non-specific reactions observed with the support-film or with the resin were eliminated by adding powdered skimmed milk or non-purified albumin into the buffers. Purified bovine serum albumin (grade V) or chicken albumin (grade III to V) were inefficient in reducing this non-specific background.     

Osmolarity of osmium tetroxide and glutaraldehyde fixatives

Synopsis The evidence available to date for the importance of fixative osmolarity is considered together with some observations on the volume changes of crab axons after fixation by osmium tetroxide and glutaraldehyde. The results obtained are compared with those obtained from crab axons and from amphioxus skin cells which had been processed and examined with the electron microscope after initial fixation in fixatives of different composition. It is concluded that the osmolarity of the fixative vehicle is of considerable importance when the fixing agent is glutaraldehyde but is of less importance when the fixing agent is osmium tetroxide or a mixture of the two agents.Preliminary observations upon crab axons fixed with glutaraldehyde in a vehicle approximating to the internal composition of the cells suggest that this approach to the design of fixative vehicles may be useful.     

Elucidation of the mechanism and end products of glutaraldehyde crosslinking reaction by X-ray structure analysis

Glutaraldehyde has been used for several decades as an effective crosslinking agent for many applications including sample fixation for microscopy, enzyme and cell immobilization, and stabilization of protein crystals. Despite of its common use as a crosslinking agent, the mechanism and chemistry involved in glutaraldehyde crosslinking reaction is not yet fully understood. Here we describe feasibility study and results obtained from a new approach to investigate the process of protein crystals stabilization by glutaraldehyde crosslinking. It involves exposure of a model protein crystal (Lysozyme) to glutaraldehyde in alkaline or acidic pH for different incubation periods and reaction arrest by medium exchange with crystallization medium to remove unbound glutaraldehyde. The crystals were subsequently incubated in diluted buffer affecting dissolution of un-crosslinked crystals. Samples from the resulting solution were subjected to protein composition analysis by gel electrophoresis and mass spectroscopy while crosslinked, dissolution resistant crystals were subjected to high resolution X-ray structural analysis. Data from gel electrophoresis indicated that the crosslinking process starts at specific preferable crosslinking site by lysozyme dimer formation, for both acidic and alkaline pH values. These dimer formations were followed by trimer and tetramer formations leading eventually to dissolution resistant crystals. The crosslinking initiation site and the end products obtained from glutaraldehyde crosslinking in both pH ranges resulted from reactions between lysine residues of neighboring protein molecules and the polymeric form of glutaraldehyde. Reaction rate was much faster at alkaline pH. Different reaction end products, indicating different reaction mechanisms, were identified for crosslinking taking place under alkaline or acidic conditions.     

Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and "postfixation" in uranyl acetate

Human leukocytes in suspension or in monolayer cultures have been processed for electron microscopy by fixation in a freshly made cold mixture of glutaraldehyde and osmium tetroxide and by "postfixation" in uranyl acetate. Simultaneous exposure to glutaraldehyde and osmium tetroxide eliminates many of the shortcomings seen when either of these agents is used alone as the initial fixative. Specimens are processed to the stage of dehydration as single cell suspensions or as very small clumps to assure rapid penetration of fixatives and efficient washing. The technique is rapid and reproducible. Electron micrographs presented in this report illustrate the ultrastructural features of human white cells prepared by this method.     

HISTOLOGICAL STAINING OF LIPIDS FOR THE LIGHT AND ELECTRON MICROSCOPE

1. It is generally agreed that the blackening of osmium tetroxide by unsaturated lipid is too unpredictable to demonstrate lipid in tissues.
2. At neutral pH osmium tetroxide combines with the double bonds in the lipoproteins of cellular membranes (mitochondria, etc.) and the deep colour reaction of ethyl gallate with this osmium provides good staining of lipid for the light microscope.
3. Osmium taken up by tissue proteins at neutral pH is only a small fraction of that taken up by the lipid. (After acid fixatives osmium tetroxide is a general protein stain.)
4. The uptake of Sudan black B by partition from dilute solution is a specific test for lipid, but in normally fixed tissue most of the structural lipid is 'bound' and is not accessible to the dye.
5. Cautious treatment of fixed tissue with dilute sodium hypochlorite will unmask this lipid for viewing by the light microscope.
6. Direct fixation with neutral osmium tetroxide is an effective method for visualizing lipid for the electron microscope (as in the ethyl gallate method for the light microscope). But the poor penetration of osmium limits its use in this way.
7. After formol/glutaraldehyde fixation much of the lipid in the tissues is 'bound' and does not take up osmium. It can be unmasked by a saturated aqueous solution of thymol.
8. The unmasked lipid can then be rendered more osmiophil by partition in a solution of the highly unsaturated terpene farnesol, thus increasing the uptake of osmium in a renewed application.
9. Some of the novel observations on tissue lipids made by these methods are reviewed.     

Swelling of golgi apparatus cisternae in monensin-treated tissues is modulated by fixation conditions

Summary Swelling of Golgi apparatus cisternae is reported to be a common response to the ionophore, monensin. However, the amount of swelling depends on fixation, thus raising the question of whether the swelling response is due to monensin or to the fixation protocol. To resolve this problem, maize root cap cells were treated with monensin and then fixed with glutaraldehyde and osmium tetroxide (applied sequentially), osmium tetroxide alone, or aqueous potassium permanganate, or were quick frozen in liquid propane and substituted in acetone-osmium tetroxide. The chemical fixatives (which take minutes to stabilize tissue elements) were judged by comparison with freeze substitution which requires only fractions of a second to stabilize tissue elements. The results verify that monensin causes cisternal swelling and that this swelling is best observed at the ultrastructural level by fixation in glutaraldehyde/osmium tetroxide or by freeze substitution.     

Cyto- and histochemical demonstration of lignins in plant cell walls: an evaluation of the chlorine water/ethanolamine-silver nitrate method of Coppick and Fowler

Summary The chlorine water/ethanolamine-silver nitrate method introduced by Coppick and Fowler for the detection of lignins was evaluated for cyto- and histochemical work using different reagents and fixatives for specimens embedded in epoxy resin. Fixation schedules tested included ethanol, glutaraldehyde, and glutaraldehyde followed by O_sO₄ as a post-fixative. Chlorine water, sodium hypochlorite, and calcium hypochlorite were the oxidising agents evaluated for their efficacy as part of the Coppick and Fowler procedure. The Coppick and Fowler method was tested against stem woody tissue ofLophomyrtus obcordata, and haustorial xylem tissue of the sucker of its attached dwarf mistletoeKorthalsella lindsayi. The presence of lignins in walls of these cells was indicated in thin sections for transmission electron microscopy by fine electron-dense deposits. Post-staining thin sections did not affect the lignin reaction, but tended to mask its effect due to increased wall contrast. In histological preparations lignified walls stained orange/brown. Counter-staining in methylene blue/azur B caused lignified walls to appear dark green/brown and non-lignified walls blue. Fixation in either ethanol or glutaraldehyde produced identical staining for lignins. Penetration by chlorine water was sometimes irregular, more so with glutaraldehyde fixation, with parts of tissues consequently not responding to the lignin reaction. Post-fixation in osmium tetroxide following primary fixation in glutaraldehyde slightly improved penetration of chlorine water. However, osmium caused greater amounts of extraneous stain deposits compared with other fixative regimes. Chlorine water was confirmed as the most effective oxidising agent for reacting with groups in lignins to produce reducing residues in the Coppick and Fowler method. Sodium hypochlorite caused no reaction. Calcium hypochlorite exhibited limited oxidative capacity resulting in slight staining for lignins. The Coppick and Fowler procedure was concluded to be a suitable method for demonstrating lignins in cyto- and histochemical preparations using material fixed in either ethanol or glutaraldehyde, and with embedding in epoxy resin.     

Effects of different fixative solutions on labeling of Concanavalin-A receptor sites in human T-lymphocytes

Summary We have studied the effect of several fixative solutions on the number of Concanavalin-A·(Con-A) receptor sites of human peripheral blood T-lymphocytes. Cells treated with different fixative solutions (glutaraldehyde (G); formaldehyde (F);G+F; osmium tetroxide (Os); Os+G; Os+F; and Os+G+F) were labeled with a Con-A gold labeled horseradish peroxidase (HRP) complex and the number of gold particles on the lymphocytic surface was evaluated. Comparison of cells treated with the different fixatives used showed significant differences in the density of labeling. After G fixation the number of gold particles was lower than after fixation with Os or F. Moreover, G used in combination with F or Os reduced the labeling obtained when the two latter fixatives were used alone.     

Immunocytochemical demonstration of LH-RH, somatostatin, and ACTH-like peptide in osmium-postfixed, resin-embedded median eminence

In guinea pig median eminences that were fixed with a glutaraldehyde paraformaldehyde mixtures, postfixed with osmium tetroxide, and embedded in araldite, immunocytochemical stainings for luteinizing hormone-releasing hormone (LH-RH), somatostatin, and alpha 17-39 ACTH have been obtained on semithin sections. These positive reactions were obtained after araldite was removed with sodium methoxide and after treatment with hydrogen peroxide. These data suggest that osmium tetraoxide postfixation can be used for the detection of low molecular weight peptides such as LH-RH, somatostatin, and ACTH-like-peptides not only in the median eminence but also in the hypothalamus.     

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bovine serum albumin oligomers produced by lipid peroxidation.

The oligomers of bovine serum albumin were produced by controlled reaction with peroxidizing linoleic acid to examine their possible utility as calibration proteins insodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polymerization was effected in reaction mixtures containing linoleic acid undergoing peroxidation in the presence of ascorbic acid, and conditions that yield soluble oligomers with a wide molecular weight distribution were established. The interaction of these soluble oligomers with sodium dodecyl sulfate exhibited a binding isotherm indistinguishable from that obtained with bovine serum albumin. Furthermore, sodium dodecy sulfate-polyacrylamide gel electrophoresis of the albumin oligomers conformed to the empirical relation of molecular weight to mobility that pertains to the use of these oligomers as standard molecular weight markers.     

FINE STRUCTURE OF THE MEDIAL NUCLEUS OF THE TRAPEZOID BODY OF THE BAT WITH SPECIAL REFERENCE TO TWO TYPES OF SYNAPTIC ENDINGS

The medial nucleus of the trapezoid body has been studied electron microscopically in two species of bat, Miniopterus schreibersi fuliginosus and Vespertilio superans, which were perfused with three different kinds of fixatives, osmium tetroxide, glutaraldehyde, and formaldehyde. Two types of synaptic endings are observed in the nucleus: the abundant calyciferous endings and the less frequently occurring "small-vesicle endings." The former endings vary greatly in size, and contain extended extracellular spaces between pre- and post-synaptic membranes. The latter endings are always small, without the extended extracellular spaces, and tend to lie side by side. In all of the materials perfused with three different fixatives, synaptic vesicles in the calyciferous endings are round in shape and larger than those in the small-vesicle endings. The shape of vesicles in the small-vesicle endings varies according to the kinds of fixatives used; round in osmium tetroxide-fixed materials, flattened in formaldehyde-fixed materials, and somewhat round or flattened in glutaraldehyde-fixed materials. It is suggested that the calyciferous endings are excitatory in nature and that the small-vesicle endings are inhibitory.