An improved method for perfusion fixation of neural tissues for electron microscopy

A method employing vascular perfusion for improved preservation of biological ultrastructure is described, and its effectiveness demonstrated for mammalian nervous tissues. Following a physiological saline flush into the aorta, hydrogen peroxide and glutaraldehyde in phosphate buffer are perfused. After buffer rinses, tissue blocks are postfixed in osmic acid and potassium ferrocyanide. The success rate is enhanced greatly by close attention to details of perfusion technique. Advantages of the method include more uniform and complete preservation. In particular, superior images of membranous elements, glycogen granules and basal laminar material are achieved. Adjustments in osmolality may render the procedure suitable for non-mammalian forms and other tissues.     

A fixation method for visualization of yeast ultrastructure in the electron microscope

A primary fixative containing glutaraldehyde (3%), acrolein (1.5%), and paraformaldehyde (1.5%) buffered in 0.05 M sodium cacodylate at pH 7.2 was applied to the cells ofCryptococcus vishniacii for 2 hours on ice. The cells were then treated with a 6% aqueous solution of potassium permanganate for 1 hour at room temperature. This method preserves most of the yeast cell fine structural components including cell walls and membrane, nuclear membrane, mitochondria, endoplasmic reticula, microbodies, vacuoles, nucleoli, and ribosomes. However, it leads to disruption of capsular materials and loss of some of the lipid and glycogen granules.This study was supported by a National Aeronautics and Space Administration (NASA) research grant NAGW-26.     

An improved method for light- and electron microscopial studies of nematode/fungal interactions

A method is presented that enables studies to be made of single nematode-fungal interactions under conditions where fungal growth at the expense of external nutrients is prevented. The nematophagous fungus Arthrobotrys ologospora was used as a model organism in these studies. The method is based on removal of the traps from the vegetative mycelium, immediately after a nematode was captured and transfer of the trap with the captured nematode into a droplet of sterile distilled water placed in a moisture chamber. In the absence of external nutrients, such isolated traps of A. oligospora were fully effective in penetrating and subsequently digesting the captured nematode. Solely vegetative mycelium was formed at the expense of the digested nematode; this developed from the trap that originally had captured the nematode. One advantage of the present method is that studies on various stages of the nematode-fungal interaction can now be performed under conditions that exclude major influences of external nutrients which otherwise could be communicated to the trap cells by way of the vegetative mycelium.     

An improved method for the inhibition of endogenous peroxidase non-deleterious to lymphocyte surface markers. Application to immunoperoxidase studies on eosinophil-rich tissue preparations

Summary The inhibitory effect of phenylhydrazine and azide combined with either pre-formed or nascent hydrogen peroxide H₂O₂ upon endogenous peroxidatic activity, expressed by tissue eosinophils in different disease states, was investigated. It was found that whilst endogenous peroxidatic activity due to eosinophils in a Hodgkin's disease and a histiocytosis X case were adequately inhibited by phenylhydrazine combined with pre-formed or nascent H₂O₂, the eosinophils in theOnchocerca volvulus nodule were either not at all or only partly inhibited by the two regimens. On the other hand, a combination of azide with nascent H₂O₂ proved consistently effective against this resistant form of endogenous peroxidatic activity. Using human tonsil sections this protocol was shown to be non-deleterious to T₄(CD4), T₆(CD1) and T₈(CD8) lymphocyte surface antigens as evidenced by the application of a standard indirect immunoperoxidase technique and the relevant monoclonal antibodies.     

Immunocytochemical detection of ricin. II. Further studies using the immunoperoxidase method

Summary Radio-iodinated ricin was injected into rat musclein vivo to establish the distribution of the toxin at various time intervals after injection. Injection site muscle and para-aortic lymph nodes were selected for localization of ricin by the immunoperoxidase technique. Sections of snap-frozen tissues were fixed using a variety of methods to establish the best protocol for the immunodetection method. This was found to be with an ether—ethanol mixture. Ricin was detected in tissue at the site of injection taken from rats sacrificed 1, 4, 8 and 24 h after injection and in tissue from animals dying from ricin intoxication after about 30 h. This method, however, failed to demonstrate unequivocally the presence of ricin in lymphoid tissue which had been indicated by the radiotracer study. The significance of these findings and their relevance to forensic diagnosis are discussed.     

Mallomonas transsylvanica,spec. nova (Chrysophyceae), light and electron microscopical studies

The new speciesMallomonas transsylvanica is described in detail. Its silica armour has been examined by light and electron microscopy. Differences and possible relationships with other species are discussed.     

The coating of mouse myocardial cells. A cytochemical electron microscopical study.

The coating of mouse myocardial cells has been investigated with a variety of cytochemical methods. The coating of the surface membrane gives a positive reaction with ruthenium red, colloidal thorium, phosphotungstic acid (PTA) at low pH, silver methenamine after periodic oxidation (PA-silver technique) and with silver proteinate after periodic oxidation and thiocarbohydrazide treatment (PA-TCH-silver technique). The coating of the T system gives almost similar results. The nexuses do not react with PTA nor with the PA-silver and PA-TCH-silver techniques, but they are strongly stained with ruthenium red which reveals periodic structures in their gaps. The specificities of the colloidal thorium technique and PAT staining have been tested by chemical treatments (methylation, acetylation, saponification), enzymatic digestions (pronase, trypsin, hyaluronidase, neuraminidase) and carbohydrate extractions (with 0.1 N NaOH and 0.05 M H2SO4). These cytochemical data indicate, considering the specificity of the reactions, that the coating of the membrane surface and the T system contains polyanionic groups. A part of them, at least, would belong to a carbohydrate-containing material (glycoproteins), whereas at the level of nexuses the sugar residues would probably be absent.     

A hot aldehyde-peroxide fixation method for electron microscopy of the free-living nematode Caenorhabditis elegans

An improved method for the fixation of the third and fourth larval stages and adults of Caenorhabditis elegans has been developed. Worms are placed in a mixture of 1.5% paraformaldehyde and 1.0% glutaraldehyde at pH 7.0 and 70 C and the suspension promptly cooled in a water bath at 20 C for 1 hr. The fixed worms are then immersed in a mixture of 5% glutaraldehyde and hydrogen peroxide at 4 C for 1 hr, stained en bloc in uranyl acetate, and embedded in resin for electron microscopy. The procedure results in superior fixation, particularly of microfilaments and microtubules. The high temperature of the initial fixation straightens the worms and thus facilitates serial sectioning.     

An improved method for covalent attachment of antibody to liposomes

A rapid and simple method is described for the incorporation of monoclonal antibody coupled with palmitic acid into liposomes prepared by the reverse-phase evaporation method (Szoka, F. and papahadjopoulos, D. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4194–4198). Palmitoyl antibody in 0.15% deoxycholate is added to a liposome suspension after the majority of the organic solvent has been removed by evaporation. Efficient incorporation (over 80%) of palmitoyl antibody occurred without leakage of the encapsulated drug. Native, unmodified antibody did not incorporate under identical conditions. About 50% of the incorporated antibodies could be readily digested by protease, while most of an internal protein marker was not, suggesting that about half of the antibodies were exposed on the outer surfaces of liposomes. Target-specific binding of antibody-liposomes has also been demonstrated in vitro with the RDM-4 lymphoma cells. This method offers a rapid and highly efficient attachment of functional antibody molecules to liposomes with high capture efficiency of drugs, and therefore should be useful in target-specific delivery of drugs mediated by liposomes.     

A modified method for studies of electron transport system activity in freshwater sediments

A method for measuring respiratory electron transport system activity (ETSA) in freshwater sediments is presented. It is a modification of the methods used for marine sediments by Christensen & Packard (1977) and Olànczuk-Neyman & Vosjan (1977). The assay is based on the reduction of the electron acceptor 2-(p iodophenyl) 3 (p nitrophenyl) 5 phenyl tetrazolium chloride (INT) by cell-free homogenates of sediment samples. This study shows that the method is sensitive, has good accuracy and can be used for freshwater sediments. The accuracy decreases at high turbidity and as yet has only been tested for sediments of the gyttja type. Untreated samples can be stored at low temperature for several weeks without any loss of activity. ETSA decreases with the depth of sediment and with increased amount of allochthonous material in the sediment.     

A gentle fixation and permeabilization method for combined cell surface and intracellular staining with improved precision in DNA quantification.

A method was developed for gentle fixation of mammalian cells and permeabilization of their membranes. The method is useful for staining of intracellular antigens or quantification of DNA content simultaneously with cell surface staining. Cells are treated for 1 h at 4 degrees C with 0.25% buffered paraformaldehyde then for 15 min at 37 degrees C with 0.2% Tween 20 detergent in PBS. The procedure permits excellent staining of intracellular proteins, very low coefficients of variation (CV) on the G0G1-peak of DNA distributions, and preservation of the integrity of cell surface antigens. The low vs. 90 degrees angle light scatter profile of cell clusters is maintained thereby allowing discrimination of different cell populations including human peripheral blood lymphocytes and monocytes for gating and analytic purposes. The method was successfully used on a variety of other cell types, including human thymocytes, murine thymocytes and spleen cells, and several leukemic cell lines. Dual-color surface antigen staining combined with DNA staining with 7-amino-actinomycin D (7-AAD) on peripheral blood mononuclear cells (PBMC) cultured with tetanus toxoid allowed the determination of the cell subset that was preferentially stimulated. Staining for internal antigens was done on CCRF-CEM for expression of CD3 epsilon and on NALM-6 for expression of mu. The technique we developed gave bright and specific staining of internal antigens in the examples presented here. It is particularly suited for correlations of internal antigen staining with DNA staining and/or surface immunofluorescence.     

A simple method to test condoms for penetration by viruses.

A method by which virus penetration through condoms can be tested with simple, inexpensive equipment is described. The method uses chi X174 bacteriophage as the challenge virus and physiologically relevant pressure. Penetration by 0.1 microliters (or less) of challenge suspension can be readily detected. As examples, latex and natural-membrane condoms were examined.