Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa

Microtubules are important in plant growth and development. Localizing microtubules in sectioned material is advantageous because it allows any tissue of interest to be studied and it permits the positional relations of the cells within the organ to be known. We describe here a method that uses semi-thin (0.5–2 m) sections of material embedded in butyl-methylmethacrylate, to which 10 mM dithiothreitol was added. After removing the embedding material and using indirect immunofluorescence staining, we obtain clear images of microtubules, actin microfilaments, callose and pulse-fed bromodeoxyuridine. This method works on the root tissues of Arabidopsis thaliana(L.) Heynh, Pinus radiataD. Don, Zamia furfuraceaAit., Azolla pinnataR. Br. and on sporophytic tissues of Funaria hygrometricaHedw. In general, most of the cells in the organs studied are successfully stained. Using this method, we find that interphase meristematic cells in all of these species have microtubules not only in the usual cortical array but also throughout their cytoplasm. The presence of the calcium chelator ethylene glycol-bis(-aminoethyl ether)N,N,N,N-tetraacetic acid EGTA in fixation buffers led to some tissue damage, and did not enhance the preservation of microtubules. The common assumption that EGTA-containing buffers stabilize plant microtubules during fixation appears unwarranted.Abbreviations BrdU 5-bromodeoxyuridine - DTT dithiothreitol - EGTA ethylene glycol-bis(-aminoethyl ether) - N,N,N,N tetraacetic acid We thank Ann Cork for technical assistance, Professor B.E.S. Gunning (Australian National University) and Drs. A.R. Hardham (A.N.U.) and R.E. Williamson (A.N.U.) for intellectual and material support, Dr D. McCurdy (A.N.U.) for the purified anti-actin antibody, and Professor B. Stone (La Trobe University, Melbourne, Australia) for generously providing the anti-callose antibody. We also thank the Electron Microscopy Unit of A.N.U. for the use of facilities. L.C.F. gratefully acknowledges financial support from the National Sciences and Engineering Research Council of Canada.     

Species differences in the negative inotropic effect of acetylcholine and soman in rat, guinea pig, and rabbit hearts.

1. Acetylcholine reduced atrial contractions by 82.5% in guinea pig, 50.8% in rat, and 41.5% in rabbit. 2. The EC50 values for the negative inotropic effect of acetylcholine were 3.3 x 10(-7) M in rat and guinea pig atria and 4.1 x 10(-6) M in rabbit atria. 3. There was no correlation between the species differences in the negative inotropic effect of acetylcholine in atria and the density or affinity of acetylcholinesterase or muscarinic receptors. 4. Inhibition of atrial acetylcholinesterase with soman reduced the EC50 of acetylcholine three-fold in all species, but did not change the maximal inotropic effect of acetylcholine. 5. Species differences in the negative inotropic effect of acetylcholine may be caused by differences in the coupling between myocardial muscarinic receptors and the ion channels that mediate negative inotropy.     

Immunocytochemical localization of Na+, K+-ATPase in the rat kidney

To determine if rat kidney Na+, K+-ATPase can be localized by immunoperoxidase staining after fixation and embedding, we prepared rabbit antiserum to purified lamb kidney medulla Na+, K+-ATPase. When sodium dodecylsulfate polyacrylamide electrophoretic gels of purified lamb kidney Na+, K+-ATPase and rat kidney microsomes were treated with antiserum (1:200), followed by [125I]-Protein A and autoradiography, the rat kidney microsomes showed a prominent radioactive band coincident with the alpha-subunit of the purified lamb kidney enzyme and a fainter radioactive band which corresponded to the beta-subunit. When the Na+, K+-ATPase antiserum was used for immunoperoxidase staining of paraffin and plastic sections of rat kidney fixed with Bouin's, glutaraldehyde, or paraformaldehyde, intense immunoreactive staining was present in the distal convoluted tubules, subcapsular collecting tubules, thick ascending limb of the loops of Henle, and papillary collecting ducts. Proximal convoluted tubules stained faintly, and the thin portions of the loops of Henle, straight descending portions of proximal tubules, and outer medullary collecting ducts did not stain. Staining was confined to basolateral surfaces of tubular epithelial cells. No staining was obtained with preimmune serum or primary antiserum absorbed with purified lamb kidney Na+, K+-ATPase, or with osmium tetroxide postfixation. We conclude that the basolateral membranes of the distal convoluted tubules and ascending thick limb of the loops of Henle are the major sites of immunoreactive Na+, K+-ATPase concentration in the rat kidney.     

Dynamic light scattering characterization of the detergent-free,delipidated (Ca2+ + Mg2+)-ATPase from sarcoplasmic reticulum

Dynamic light scattering studies have been conducted on the delipidated and detergent-removed (Ca²⁺ + Mg²⁺)-ATPase protein assemblies. Specific characterization of the state of aggregation and the extent of conformation change upon delipidation and detergent removal has been made. The results show that the prominent species are dimers and tetramers of very globular nature, with axial ratios of less than 2 : 1. The hydrodynamic radii of the dimers and the tetramers are, respectively, 57.5 Å and 74.5 Å.The globular nature of these observed entities differ from the delipidated ATPase proteins recently obtained (LeMaire, M., Jorgensen, K.E., Roigaard-Petersen, H. and Moller, J.V. (1976) Biochemistry 15, 5805–5812). Present results suggest that upon the removal of detergents from the lipid-free ATPase protein assembly, only a rather limited degree of aggregation takes place. Such a condition is consistent with models of the membrane protein system which has limited regions of hydrophobic contact. Oligomeric assemblies with aqueous channels is a possible active Ca²⁺ transport model consistent with results of the present data, as well as the data from several other recent studies.     

Sarcomere length dispersion in single skeletal muscle fibers and fiber bundles.

Light diffraction patterns produced by single skeletal muscle fibers and small fiber bundles of Rana pipiens semitendinosus have been examined at rest and during tetanic contraction. The muscle diffraction patterns were recorded with a vidicon camera interfaced to a minicomputer. Digitized video output was analyzed on-line to determine mean sarcomere length, line intensity, and the distribution of sarcomere lengths. The occurrence of first-order line intensity and peak amplitude maxima at approximately 3.0 mum is interpreted in terms of simple scattering theory. Measurements made along the length of a singel fiber reveal small variations in calculated mean sarcomere length (SD about 1.2%) and its percent dispersion (2.1% +/- 0.8%). Dispersion in small multifiber preparations increases approximately linearly with fiber number (about 0.2% per fiber) to a maximum of 8-10% in large bundles. Dispersion measurements based upon diffraction line analysis are comparable to SDs calculated from length distribution histograms obtained by light micrography of the fiber. First-order line intensity decreases by about 40% during tetanus; larger multifibered bundles exhibit substantial increases in sarcomere dispersion during contraction, but single fibers show no appreciable dispersion change. These results suggest the occurrence of asynchronous static or dynamic axial disordering of thick filaments, with a persistence in long range order of sarcomere spacing during contraction in single fibers.     

The fine structure of polychaete septate junctions

Summary Epidermal septate junctions of Nereis sp. and Cirriformia sp. fixed with OsO₄ or glutaraldehyde/OsO₄ display variable structure in electron micrographs. In transverse section the septa are often indistinct and obscured by opaque material that fills the junctional cleft. Septa (spaced at 180–280 Å) are more clearly defined in slightly oblique transverse section; they exhibit an electron lucent center and appear to be linked by arms. En face views of the junction show a honeycomb pattern. Cytoplasmic faces of junctional membranes are backed with plaques opposite the septa. Lanthanum used as a tracer delineates junctional structure in negative contrast. In transverse section a chain-like lattice is present in the junctional cleft. En face views show parallel rows of pleated elements often linked by arms into honeycomb arrays. Oblique sections demonstrate that these pleated elements are continuous with the chain-like lattice seen in transverse sections. Lanthanum does not pass entirely through the junction. Lanthanum reveals that the septa have a very intricate substructure, but it is difficult to visualize the architecture that could generate the various images presented by these junctions when seen in different orientations. However, it is clear that these junctions possess some features that are diagnostic of several supposedly different types of septate junctions in invertebrates.Supported by USPHS grants NIH 5 P01 NS-07512, NIH 2701 GM-00102, and NB-00840, and by a grant from the Pomona College Research CommitteeI thank Sarah Wurzelmann, Stanley Brown, Nancy Kelly, and Gerhard Ott for excellent technical assistance. Portions of this study were carried out while I was a Postdoctoral Fellow in the Department of Anatomy, Albert Einstein College of Medicine. I dedicate this article to Berta Scharrer as a token of appreciation and affection for her guidance, encouragement, inspiration, and example of excellence