ALTERATION OF THE CONFORMATION OF PROTEINS IN RED BLOOD CELL MEMBRANES AND IN SOLUTION BY FIXATIVES USED IN ELECTRON MICROSCOPY

The effects of several commonly employed fixatives on the three-dimensional conformations of two soluble proteins and the protein of intact red blood cell membranes have been studied by means of circular dichroism measurements in the spectral region of the peptide absorption bands. The fixatives used produced significant and parallel conformational changes in all of the proteins, in the increasing order: glutaraldehyde; OsO₄; glutaraldehyde followed by OsO₄; and KMnO₄. The last two treatments obliterated most of the helical character of the proteins. The significance of these observations to the preparation of specimens for electron microscopy is discussed.     

The Use of OsO4 Fixative for Feulgen-Stained Preparations

OsO₄ has many advantages over Carnoy's fixative mixture for the Feulgen nuclear staining in the protozoan Tokophrya infusionum. While Carnoy's fluid used prior to the Feulgen reaction produces shrinkage of the macronucleus and coarse clumping of its chromatin bodies, OsO₄ preserves faithfully the size and shape of the macronucleus and its chromatin material. This finding seems to be of special importance in view of the fact that electron microscopy relies on OsO₄ fixation. The satisfactory preservation of structured detail in Feulgen-stained preparations is of importance for the correlation of histochemical and morphological information.     

On the influence of fixations on the normal hydration of rabbit cornea and the total amount of acid mucopolysaccharides in the stroma

Summary The authors studied the influence of fixations on the normal hydration of the rabbit cornea and the total amount of acid mucopolysaccharides (AMPS) in the stroma. The following fixatives were used: formol-calcium chloride at 19° C for 24 hours, formolcetylpyridinium chloride (CPC) at 19 and 28° C for 48 hours, Lillie's fixation at 19° C for 24 hours and Carnoy's fluid at 19° C for 30 minutes. The sections of the corneae were stained with Alcian blue, colloidal Fe³⁺ in the modification according to Rinehart and Abu'l Haj and with toluidine and methylene blue. The amount of AMPS was determined with the method of Rondle and Morgan and the total hydration of the stroma by weighing the corneae before and after using different fixative fluids and by calculation of obtained values on dry weight.The best results were obtained by using formol-CPC at 28° C. At the ordinary room temperature (±19° C) it was the poorest fixation, however, as the corneae in this solution became hydrated. Formol-calcium chloride was the second in the row and it was much better than Lillie's and Carnoy's fluid.The amount of AMPS in the stroma was not essentially changed by the effect of fixatives. Within 24–48 hours formol-CPC at 28° C retained the normal content and formol-calcium chloride caused the 11% decrease of AMPS maximally. The loss of AMPS after other fixatives was minimal.The intensity of staining with cationic dyes in paraffin sections was different after individual fixatives and after the kind of their application and was dependent chiefly on the state of hydration of the corneal stroma: It is impossible to interpret the results of staining reactions in terms of the quantity of AMPS as it was hitherto done.     

The effect of different fixatives and length of fixation time on subsequent AgNOR staining for frozen and paraffin-embedded tissue sections

Summary The AgNOR technique has been used extensively in studies investigating the possibility that the numbers and appearances of the intranuclear structures stained are markers of malignancy. The method has the advantage of being applicable to many different types of histological material, including paraffin-embedded tissue. However, it has been suggested that the visualization of AgNORs is dependent on the type and time of fixation employed. This study set out to measure this effect with the following commonly-used fixatives: acetone, absolute ethanol, methanol, Carnoy's fluid, Bouin's fluid, 4% glutaraldehyde, 10% neutral buffered formalin and 10% formol-saline. Both frozen sections and blocks of fresh tonsil were fixed for varying times, the blocks of tissue then being processed routinely. With the frozen sections AgNORs were easier to discern than in sections of paraffin-embedded tissue, and more intranucleolar AgNORs were visible when alcoholic fixatives were used than with aldehyde fixation. The effects of different fixatives on AgNOR appearance in paraffin sections is, however, more complex. Despite the variation caused by different fixatives, AgNORs could be demonstrated adequately with all the fixatives studied. It is concluded that fixation is not a limitation to the study of AgNORs provided that the time and type of fixative is controlled.     

INFLUENCE OF GLUTARALDEHYDE AND/OR OSMIUM TETROXIDE ON CELL VOLUME, ION CONTENT, MECHANICAL STABILITY, AND MEMBRANE PERMEABILITY OF EHRLICH ASCITES TUMOR CELLS

Effects of fixation with glutaraldehyde (GA), glutaraldehyde-osmium tetroxide (GA-OsO₄), and osmium tetroxide (OsO₄) on ion and ATP content, cell volume, vital dye staining, and stability to mechanical and thermal stress were studied in Ehrlich ascites tumor cells (EATC). Among variables investigated were fixation time, fixative concentration, temperature, osmolality of the fixative agent and buffer, total osmolality of the fixative solution, osmolality of the postfixation buffer, and time of postfixation treatment in buffer (Sutherland, R. M., et al. 1967. J. Cell Physiol. 69:185.). Rapid loss of potassium, exchangeable magnesium, and ATP, and increase of vital dye uptake and electrical conductivity occurred with all fixatives studied. These changes were virtually immediate with GA-OsO₄ or OsO₄ but slower with GA (in the latter case they were dependent on fixative temperature and concentration) (Foot, N. C. 1950. In McClung's Handbook of Microscopical Technique. 3rd edition. 564.). Total fixative osmolality had a marked effect on cell volume with OsO₄ but little or no effect with GA or GA-OsO₄. Osmolality of the buffer had a marked effect on cell volume with OsO₄, whereas with GA or GA-OsO₄ it was only significant at very hypotonic buffer osmolalities. Concentration of GA had no effect on cell volume. Osmolality of the postfixation buffer had little effect on cell volume, and duration of fixation or postfixation treatment had no effect with all fixatives. Freezing and thawing or centrifugal stress (up to 100,000 g) had little or no effect on cell volume after all fixatives studied. Mechanical stress obtained by sonication showed that OsO₄ alone produced poor stabilization and that GA fixation alone produced the greatest stabilization. The results indicate that rapid membrane permeability changes of EATC follow fixative action. The results are consistent with known greater stabilizing effects of GA on model protein systems since cells were also rendered relatively stable to osmotic stress during fixation, an effect not noted with OsO₄. After fixation with GA and/or OsO₄ cells were stable to osmotic, thermal, or mechanical stress; this is inconsistent with several earlier reports that GA-fixed cells retain their osmotic properties.     

Lichtmikroskopische Untersuchungen zur Strukturerhaltung farbstoffinduzierter autophagischer Vakuolen durch verschiedene Fixantien

Zusammenfassung Fibroblastenkulturen wurden mit Mepacrin (Atebrin®), Neutralrot und Toluidinblau unter vergleichbaren Bedingungen vitalgefärbt. Die Farbstoffe induzieren die Bildung autophagischer Vakuolen (Autolysosomen) im Cytoplasma. Die Eignung von sieben verschiedenen Fixantien zur Erhaltung dieser im lichtmikroskopischen Sinn neugebildeten Strukturen wurde untersucht.Kriterien der jeweiligen Fixationsleistung waren einmal die Erhaltung der autophagischen Vakuolen an sich, zum anderen die Erhaltung ihrer farbstoffabhängigen, morphologischen Individualität.Als wenig leistungsfähig haben sich erwiesen die Lösungen nach Carnoy und Bouin sowie Formol. Glutaraldehyd bewahrt die Lysosomenstruktur befriedigend, jedoch nicht ausreichend stabil für weitere, etwa histochemische, Eingriffe. Kaliumbichromat gewährleistet bessere Stabilität, jedoch nur geringe Lebensähnlichkeit der Autolysosomen.OsO₄ und NaMnO₄ sind den anderen Fixantien hinsichtlich der Erfüllung beider Kriterien deutlich überlegen. Die Befunde werden mit dem lipidstabilisierenden Effekt, den beide Metalloxydverbindungen an den phospholipidreichen Autolysosomen ausüben, in Zusammenhang gebracht.Unterschiede in der Wirkung ließen sich nach Anwendung von OsO₄ und NaMnO₄ an den AV nachweisen: Mepacrin-AV werden durch OsO₄ etwas lebensähnlicher erhalten als durch NaMnO₄. Die Neutralrot-AV und Toluidinblau-AV mit deutlicher vakuolärer Struktur werden nur durch Permanganat im Zusammenhang erhalten, mit deutlicher Abgrenzung der Toluidinblau-induzierten von den Neutralrot-induzierten Autolysosomen.Nach Osmium- und Permanganatfixation zeigen die Zellkulturen starke Affinität zu Methylenblau, nicht Eosin. Nur die OsO₄-fixierten Autolysosomen halten gegenüber Alkoholeinwirkung ihre Anfärbung im wesentlichen bei.Die Befunde werden diskutiert.

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Light microscopical investigations on structural preservation of Dye-Induced autophagic vacuoles by diverse fixatives

Summary Fibroblasts grown in monolayer were subjected to vital staining by mepacrine (Atebrine®), neutral red and toludine blue under comparable conditions. These dyes induce the formation of autophagic vacuoles (autolysosomes) in the cytoplasm. The preservation of these structures, which are considered to be newly formed in the dimension of the light microscope, by seven different fixatives has been examined.The criteria employed to assess the performance of each fixative consisted of 1. the preservation of the autophagic vacuoles per se and 2. their dye-dependent morphological characteristics.Fixation by Carnoy's or Bouin's solution as well as by formaline gave poor results. Glutaraldehyde preserved lysosomal structure satisfactorily, but not adequately for further application of histochemical methods. Potassium dichromate has a stabilizing effect on autophagic vacuoles, however, structures are not equivalent to those observed in living cells.Osmium tetroxide (OsO₄) and sodium permanganate (NaMnO₄) are superior to the other fixatives with regard to the afore mentioned criteria. These observations are explained by the wellknown lipid-stabilizing effect which both metal oxides are expected to exert on autolysosomes with their high content of phospholipids.After fixation with OsO₄ and NaMnO₄ diverging effects on autophagic vacuoles could be ascertained. Mepacrine-induced autophagic vacuoles are preserved somewhat more accurately by OsO₄ than by NaMnO₄.Autolysosomes induced by neutral red and toluidine blue display a more vacuolated appearance and are preserved as a whole only by permanganate. Distinct differences exist between autophagic vacuoles induced by toluidine blue and those induced by neutral red.After fixation by OsO₄ and NaMnO₄ cells from culture display a strong affinity to methylene blue, but not to eosin. The staining of autolysosomes by methylene blue is resistant to ethanol after fixation in OsO₄ only.The results are discussed.

Die Arbeit wurde mit Unterstützung der Firma Hoffmann-La Roche und Co. A.G., Basel, durchgeführt.     

ELECTRON MICROSCOPE OBSERVATIONS ON FROZEN-DRIED CELLS

To explore the problem of artefacts which may be produced during usual fixation, dehydration, and embedding, the authors have examined pancreas, liver, and bone marrow frozen at about -180°C., dried, at -55 to -60°C., embedded in methacrylate, sectioned, and floated on a formol-alcohol mixture. By these treatments the labile structure of living cells can be fixed promptly and embedded in methacrylate avoiding possible artefacts caused by direct exposure to chemical fixatives. Cell structures are ultimately exposed to a fixative when the sections are floated on formol-alcohol, but at this stage artefacts due to chemical fixation are expected to be minimized, as the fixatives act on structures tightly packed in methacrylate polymer. In the central zone of tissue blocks so treated, the cells are severely damaged by ice crystallization but at the periphery of the blocks the cell structure is well preserved. In such peripherally located cells, elements of the endoplasmic reticulum (ER), Palade's granules, homogeneously dense mitochondria, and nuclear envelopes and pores, can be demonstrated without poststaining with OsO₄. The structural organization in the nucleus is distorted by vacuolization. The mitochondrial membranes and cristae, cellular membrane, and the Golgi apparatus, however, are detected only with difficulty. The Golgi region generally appears as a light zone, in which some ambiguous structures are encountered. After staining the sections with OsO₄ or Giemsa solution, an inner mitochondrial structure which resembles the cristae seen in conventional OsO₄-fixed specimens appears, but the limiting membrane is absent. Treatment with OsO₄ or Giemsa solution also renders more distinct the membrane of the ER and Palade's granules but not the Golgi apparatus and cellular membrane. Treatment with ribonuclease results in the disappearance of Palade's granules. On the strength of these observations the authors conclude that OsO₄ fixation gives a satisfactory preservation of such cell structures as the nuclear envelope, endoplasmic reticulum, and Palade's granules, though it may induce slight swelling of these cell components.     

Effects of fixation and processing on the immunohistochemical visualization of type-I, -III and -IV collagen in paraffin-embedded liver tissue

Summary We compared the effects of different fixatives and enzymatic-digestion procedures on the immunohistochemical visualization of type-I,-III and-IV collagen in paraffin-embedded normal human liver sections. None of the fixatives tested allowed the staining of these antigens without prior enzymatic digestion. The best results i.e. strong staining intensity and well-defined localization, were obtained when liver tissue was fixed in Bouin's fluid or in other solutions containing picric acid. Several other fixatives, including Carnoy's fluid, Lillie's AAF, 10% neutral formalin and 96% ethanol, gave unsatisfactory results. Pepsin was ineffective for unmasking type-I and-III collagen antigens, and was only partially effective for visualizing the type-IV collagen antigen. The best results were obtained when material fixed in Bouin's fluid was embedded in paraftin and digested with trypsin. Using this procedure, the results were comparable to those obtained in unfixed frozen sections with respect to the staining intensity, specificity and non-specific staining.     

A Detailed Analysis of the Effects of Various Fixatives on Animal Tissue with Particular Reference to Muscle Tissue

Nine different fixatives (Carnoy's, Susa, Baker's formalin, 5% formalin, 10% formalin, 10% formol saline, Bouin, Zenker, and 2.5% glutaraldehyde) were compared by two methods. Gelatin-albumin gels were used to study volum changes after fixation and after various stages of subsequent processing. The appearance and hardness of the gels were also noted. The fixatives either shrunk or swelled the gels, but dehydration and clearing shrunk the gels in all cases. Sampkes of muscle tissue from one location in beef longissimus dorsi muscle were also placed in the different fixatives and processed. Various features were noted for each fixative, including the ease with which the paraffin wax blocks were cut and the staining ability of the sections in Mallory's triple stain. The diameters of the muscle fibers were measured from transverse sections of these samples and compared with the mean diameter of muscle fibera in a frozen unfixed section of muscle tissue. It was found that the fixatives had the same shrinkage effects on both the gels and the muscle samples. Analysis of variance tests showed that the various fuatives caused different degrees of shrinkage. Statistical details are given for the amounts of shrinkage caused by each fixative. Both the general histological picture and the amount of shrinkage were considered when deciding the bcst fixative. Carnoy was found to be the best of the fixatives investigated.     

Simplified and optimized kinetochore detection: cytogenetic marker for late-G2 cells

Cytogenetic detection of kinetochore proteins using the CREST antibody coupled with secondary antibodies labeled with different fluorescent probes has been optimized for several in vitro mammalian cell lines. This study investigated selected parameters including the influence of common fixatives (methanol, ethanol, methanol: acetic acid (3:1)), detergents (Tritron-X100, Tween), fluorescent probes (CY3, BODIPY, FITC), washing protocols, and an antifading agent (paraphenylenediamine) on the detection of kinetochore proteins in control and X-ray (240 kVp)-irradiated cells. Utilizing an optimized fixation and staining protocol, a brilliant visualization of kinetochores in interphase cells was obtained in control as well as X-ray-irradiated interhase cells. Application of this improved kinetochore staining methodology readily permits discriminating cells containing either single or paired kinetochores, the latter of which are characteristic of late-G₂ phase and prophase cells.     

Side-chain conformations in 4-alpha-helical bundles.

The distribution of the chi(1), chi(2) dihedral angles in a dataset consisting of 12 unrelated 4-alpha-helical bundle proteins was determined and qualitatively compared with that observed in globular proteins. The analysis suggests that the 4-alpha-helical bundle motif could occasionally impose steric constraints on side chains: (i) the side-chain conformations are limited to only a subset of the conformations observed in globular proteins and for some amino acids they are sterically more constrained than those in helical regions of globular proteins; (ii) aspartic acid and asparagine occasionally adopt rotamers that have not been previously reported for globular or helical proteins; (iii) some rotamers of tyrosine and isoleucine are predominantly or exclusively associated with hydrophobic core positions (a, d); (iv) mutations in the hydrophobic core occur preferentially between residue types which among other physicochemical properties also share a predominant rotamer.     

Two Organic Fixatives for Acid Mucopolysaccharides

Cetyl pyridinium chloride, 0.5% in 4% aqueous formaldehyde and 5-aminoacridine hydrochloride, 0.4% in 50% aqueous ethanol, have been tested as fixatives for acid mucopolysaccharides in a variety of tissues. These solutions are superior to 4% aqueous formaldehyde, Carnoy's fluid and basic lead acetate for this purpose and also give good nuclear fixation. Tissues containing these mucopolysaccharides are particularly well defined with the acridine-ethanol fixative.     

The importance of fixation procedures on DNA template and its suitability for solution-phase polymerase chain reaction and PCR in situ hybridization

Summary Conventional solution-phase polymerase chain reaction (PCR) and in situ PCR/PCR in situ hybridization are powerful tools for retrospective analysis of fixed paraffin wax-embedded material. Amplification failure using these techniques is now encountered in some centres using archival fixed tissues. Such ailures may not only be due to absent target DNA sequences in the tissues, but may be a direct effect of the type of fixative, fixation time and/or fixation temperature used. The type of nucleic acid extraction procedure applied will also influence amplification results. This is particularly true with in situ PCR/PCR in situ hybridization.To examine these effects in solution-phase PCR, -globin gene was amplified in 100 mg pieces of tonsillar tissue fixed in Formal saline, 10% formalin, neutral buffered formaldehyde, Carnoy's, Bouin's, buffered formaldehyde sublimate, Zenker's, Helly's and glutaraldehyde at 0 to 4°C, room temperature and 37°C fixation temperatures and for fixation periods of 6, 24, 48 and 72 hours and 1 week. DNA extraction procedures used were simple boiling and 5 days' proteinase K digestion at 37°C. Amplified product was visible primarily yet variably from tissue fixed in neutral buffered formaldehyde and Carnoy's, whereas fixation in mercuric chloride-based fixatives produced consistently negative results. Room temperature and 37°C fixation temperature appeared most conducive to yielding amplifiable DNA template. Fixation times of 24 and 48 hours in neutral buffered formaldehyde and Carnoy's again favoured amplification.Fixed SiHa cells (containing 1–2 copies of HPV 16) were examined using PCR in situ hybridization for the amplification of HPV 16. Discrete and diffuse amplification signals were obtained. Neutral buffered formaldehyde fixation for 12–24 hours yielded amplifiable material suitable for use with PCR in situ hybridization. Overall amplification success within cellular preparations was 40%, with non-specific background staining also seen. Possible technical problems encountered with PCR in situ hybridization are discussed.     

Effects of fixation and processing on the immunohistochemical visualization of type-I, -III and -IV collagen in paraffin-embedded liver tissue

We compared the effects of different fixatives and enzymatic-digestion procedures on the immunohistochemical visualization of type-I, -III and -IV collagen in paraffin-embedded normal human liver sections. None of the fixatives tested allowed the staining of these antigens without prior enzymatic digestion. The best results i.e. strong staining intensity and well-defined localization, were obtained when liver tissue was fixed in Bouin's fluid or in other solutions containing picric acid. Several other fixatives, including Carnoy's fluid, Lillie's AAF, 10% neutral formalin and 96% ethanol, gave unsatisfactory results. Pepsin was ineffective for unmasking type-I and -III collagen antigens, and was only partially effective for visualizing the type-IV collagen antigen. The best results were obtained when material fixed in Bouin's fluid was embedded in paraffin and digested with trypsin. Using this procedure, the results were comparable to those obtained in unfixed frozen sections with respect to the staining intensity, specificity and non-specific staining.     

Ultracytochemical identification of catecholamine-containing vesicles in the ligated sciatic nerve of the rat. Comparison with sympathetic nerve terminals

Summary Using the fixation procedure of Tranzer, three kinds of granular vesicles were identified in certain unmyelinated fibres of rat sciatic nerves proximal to a ligature: (1) small vesicles (SGV: 30–60 nm in diameter), (2) large vesicles (LGV: 60–100nm in diameter), and (3) large elongated vesicles (LEV: 60–100nm in diameter). A comparative study concerning the distribution of these granular vesicles was carried out using a cytopharmacological method (reserpine) and employing different fixatives (aldehydes + OsO₄, or OsO₄ alone) in periarterial nerve plexus of the femoral artery, vas deferens and the pineal organ.Use of Tranzer's method allows preservation in almost all granular vesicles of a strongly electron-dense core, while with the other fixatives mainly small, eccentric dense cores occur in the vesicles. Two main features were observed in ligated sciatic nerves: (i) a clear increase in the number of LGV, and (ii) the presence of LEV, considered as a variety of LGV rather than a new population of granular vesicles. Reserpine caused the cores of SGV to disappear almost completely, while LGV and LEV remained only partly depleted. The original method combining Tranzer's fixation procedure with radioautography revealed radioautographic labelling only in the unmyelinated fibres of ligated sciatic nerves and mainly superimposed over SGV, LGV and LEV. It is suggested that (i) SGV, LGV and also LEV represent possible storage sites of catecholamines, and (ii) a local morphogenesis of SGV from the large vesicles occurs in ligated sympathetic nerve fibres.     

Improved immunohistochemical localization of tissue antigens using modified methacarn fixation

Detailed comparative studies have been carried out using a wide range of aqueous and nonaqueous based fixatives in order to select the optimal fixation schedule for the immunohistochemical localization of four antigens: type IV collagen, actin, Factor VIII, and epithelial membrane antigen (EMA). Modified methacarn fixation provides an ideal combination of maximum staining and morphological preservation for these antigens. Enhanced immunoreactivity with this fixative was also noted for a broad spectrum of antigens, including myoglobin, glial fiber acidic protein, keratin, myosin, laminin, prostatic acid phosphatase, alpha-fetoprotein, and carcinoembryonic antigen. Although it is not recommended for most cell surface antigens, this fixative may have a definite place in diagnostic surgical pathology.     

Effects of fixation, dehydration and staining on dimensions of myxosporidan

The effects of fixation, dehydration and staining on the morphological dimensions of myxo- and microsporidan spores were tested. Seven fixatives, two dehydrants and five stains were tested. Ten % formalin produced the least shrinkage and provided the best cytological detail of fixed material in both types of spores. All fixatives caused shrinkage of myxosporidan spore length and polar capsule length. Spore capsule width and polar capsule width were unaffected by 10% formalin. Ethyl alcohol caused no significant change in spore width. Microsporidan spore length shrunk with all fixatives, but spore width was generally unaffected. Dehydration, with either isopropyl alcohol or acetone, produced additional, significant shrinkage. The influence of stains on spore size was negligible. Heidenhains iron hematoxylin followed by eosin, and Mallory's analine-blue collagen stain, effectively stained myxo- and microsporidan spores.     

Die Feinstruktur der interstitiellen Grundsubstanz im inneren Nierenmark der Ratte bei verschiedenen Fixierungsarten

Zusammenfassung Das Interstitium des inneren Nierenmarkes von Ratten wurde nach Anwendung verschiedener Fixierungsarten und Versuchsanordnungen elektronenmikroskopisch untersucht. Neben Bindegewebszellen und kollagenen Fibrillen enthält der interstitielle Raum bandförmige, basalmembranartige Strukturen, in deren weiträumigen Maschen zwei weitere Komponenten, nämlich ein feingranuläres und feinfilamentäres Material sowie kontrastreiche, globuläre Partikel liegen. Diese beiden Formelemente stellen sich bei den verschiedenen Fixierungsarten und Versuchsanordnungen unterschiedlich dar. Bei der feingranulären und feinfilamentären Komponente handelt es sich wahrscheinlich um interstitielles Blutplasma. Diese Deutung wird gestützt durch die morphologische Ähnlichkeit mit dem Blutplasma der Blutgefäße, die unterschiedliche Darstellbarkeit nach Glutardialdehyd-und Formalinfixierung sowie durch die Befunde nach Gefäßperfusion und Nierenvenenstauung. Das Verhalten der globulären Partikel nach OsO₄-Einwirkung steht in Einklang mit der schon früher gegebenen Interpretation als präzipitierte saure Mukopolysaccharide. Ihr elektronenoptisches Bild wird im Vergleich zur bekannten makromolekularen Struktur der Mukopolysaccharid-Protein-Moleküle diskutiert.

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The ultrastructure of the interstitial ground substance in the inner medulla of the rat kidney investigated after various fixations

Summary The interstitial ground substance in the inner medulla of the rat kidney is examined electron microscopically after fixation with various fixation agents and under different experimental conditions. The papillary interstitium contains besides interstitial cells and few collagen fibrils basement membrane like material and two other components, fine granular-fibrillar elements and globular particles with heavy contrast. The appearance of the two latter interstitial components varies according to the different fixation agents used and to the experimental conditions. The fine granular and fine fibrillar material surrounds the globular particles numerously after glutardialdehyde-fixation but scarcely after formaldehyde-fixation. It increases after renal vein occlusion, but is diminished or absent, if the kidney is rinsed bloodfree. These results allow to conclude that this material represents interstitial bloodplasma. The behaviour of the globular particles after OsO₄-fixation agrees well with the earlier interpretation according to which they represent precipitated acid mucopolysaccharides. Their electron microscopic appearance is discussed in comparison with the known macromolecular structure of the acid mucopolysaccharides.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

The importance of tissue fixation for light microscopic immunohistochemical localization of peroxisomal proteins: the superiority of Carnoy's fixative over Baker's formalin and Bouin's solution

We have compared the effects of fixation with three commonly used fixatives upon preservation of the antigenicity of six peroxisomal proteins in rat liver using both immunohistochemical staining and Western blotting of fixed tissue extracts. The immunoreactivity of all six peroxisomal proteins was well preserved and peroxisomes were clearly identified in material fixed in Carnoy's fixative. Moreover, the corresponding proteins stained well in Western blots prepared from extracts of Carnoyfixed material. The intensity of the immunohistochemical staining was reduced at different rates for individual peroxisomal proteins after fixation in Baker's formalin, but peroxisomes were still well visualized with antibodies to catalase and some -oxidation enzymes. No evidence of immunohistochemical staining for any peroxisomal antigens was obtained after fixation in Bouin's fluid. For detection of the antibody binding sites in Carnoy's fixed material, the avidin-biotin-peroxidase complex (ABC) with aminoethyl carbazole as chromogen was found to be superior to the methods of peroxidase-antiperoxidase/diaminobenzidine and protein A-gold with silver intensification. Using Carnoy-fixative and the ABC-method, we demonstrate light microscopic immunohistochemical localization of peroxisomal antigens in several rat tissues as well as in human post-mortem liver.