Isolation of leaf bundle sheath protoplasts from C4 dicot species and intracellular localization of selected enzymes

Renal hypertrophy was induced in 100 g male rats by the injection of 250 mg folic acid (FA) dissolved in 0.3 M NaHCO3/kg body weight (i.v.). Preliminary studies of the biochemical alterations in ribonucleic acid (RNA) metabolism of the renal tissue have been reported recently (1). They are: RNA content and concentration, orotic acid-c14 incorporation into RNA and acid soluble nucleotide pool, intracellular localization of the newly synthesized RNA, and the specific activity of enzymes of the de novo pyrimidine biosynthesis pathway. The present report describes the light and electron microscopic observations in these animals. For light microscopy, kidney slices were fixed in formalin, embedded, sectioned, and stained with H & E and PAS.

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The Immuno-Electron Microscopic Localization of the Mo-Fe Protein Component of Nitrogenase in Cells of Azotobacter Vinelandii

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100073313 ◽

1973 ◽

Vol 31 ◽

pp. 574-575

Author(s):

J. T. Stasny ◽

R. C. Burns ◽

R. W. F. Hardy

Keyword(s):

Cellular Localization ◽

Direct Evidence ◽

Azotobacter Vinelandii ◽

Intracellular Localization ◽

Protein Component ◽

Electron Microscopic ◽

Thin Sections ◽

Fe Protein ◽

Intracytoplasmic Membranes

Structure-functlon studies of biological N2-fixation have correlated the presence of the enzyme nitrogenase with increased numbers of intracytoplasmic membranes in Azotobacter. However no direct evidence has been provided for the internal cellular localization of any nitrogenase. Recent advances concerned with the crystallizatiorTand the electron microscopic characterization of the Mo-Fe protein component of Azotobacter nitrogenase, prompted the use of this purified protein to obtain antibodies (Ab) to be conjugated to electron dense markers for the intracellular localization of the protein by electron microscopy. The present study describes the use of ferritin conjugated to goat antitMo-Fe protein immunoglobulin (IgG) and the observations following its topical application to thin sections of N2-grown Azotobacter.

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An immunocytochemical study of maternal immunoglobulin localization in the suckling pig intestine

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010016217x ◽

1990 ◽

Vol 48 (3) ◽

pp. 922-923

Author(s):

László G. Kömüves ◽

Donna S. Turner ◽

Kathy S. McKee ◽

Buford L. Nichols ◽

Julian P. Heath

Keyword(s):

Sodium Ethoxide ◽

Intracellular Localization ◽

Protein A ◽

Tween 20 ◽

Intestinal Epithelial ◽

Immunocytochemical Study ◽

Fish Skin ◽

Newborn Piglets ◽

Rabbit Igg ◽

Fish Skin Gelatin

In this study we used colloidal gold probes to detect the intracellular localization of colostral immunoglobulins in intestinal epithelial cells of newborn piglets.Tissues were obtained from non-suckled newborn and suckled piglets aged between 1 hour to 1 month. Samples were fixed in 2.5 % glutaraldehyde, osmicated and embedded into Spurr’s resin. Thin (80 nm) sections were etched with 5% sodium ethoxide for 5 min, washed and treated with 4 % sodium-m-periodate in distilled water for 30 min. The sections were then first incubated with blocking buffer (2 % BSA, 0.25 % fish skin gelatin, 0.5 % Tween 20 in 10 mM Trizma buffer, pH=7.4 containing 500 mM NaCl) for 30 min followed by the immunoreagents diluted in the same buffer, 1 hr each. For the detection of pig immunoglobulins a rabbit anti-pig IgG antiserum was used followed by goat anti-rabbit IgG-Au10 or protein A-Au15 probes.

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Immunoferritin Labelling of Murine Leukemia Virus Antigens in thin Sections

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600002221 ◽

1980 ◽

Vol 38 ◽

pp. 508-509

Author(s):

Ray A. Weigand ◽

Gregory C. Varjabedian

Keyword(s):

Murine Leukemia Virus ◽

Leukemia Virus ◽

Intracellular Localization ◽

Uranyl Acetate ◽

Antibody Technique ◽

Thin Sections ◽

Tumor Virus ◽

Labelling Procedure ◽

Unlabelled Antibody ◽

Murine Leukemia

We previously described the intracellular localization of murine mammary tumor virus (MuMTV) p28 protein in thin sections (1). In that study, MuMTV containing cells fixed in 3% paraformaldehyde plus 0.05% glutaraldehyde were labelled after thin sectioning using ferritin-antiferritin in an unlabelled antibody technique. We now describe the labelling of murine leukemia virus (MuLV) particles using the unlabelled antibody technique coupled to ferritin-Fab antiferritin. Cultures of R-MuLV in NIH/3T3 cells were grown to 90% confluence (2), fixed with 2% paraformaldehyde plus 0.5% glutaraldehyde in 0.1 M cacodylate at pH 7.2, postfixed with buffered 17 OsO4, dehydrated with a series of etha-nols, and embedded in Epon. Thin sections were collected on nickel grids, incubated in 107 H2O2, rinsed in HEPES buffered saline, and subjected to the immunoferritin labelling procedure. The procedure included preincubation in 27 egg albumin, a four hour incubation in goat antisera against purified gp69/71 of MuLV (3) (primary antibody), incubation in F(ab’)2 fragments of rabbit antisera to goat IgG (secondary antibody), incubation in apoferritin, incubation in ferritin-Fab ferritin, and a brief fixation with 2% glutaraldehyde. The sections were stained with uranyl acetate and examined in a Siemens IA electron microscope at an accelerating voltage of 60 KV.

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