A double staining technique for improved contrast of thin sections from Spurr-embedded tissue

1978 ◽  
Vol 56 (1) ◽  
pp. 129-132 ◽  
Author(s):  
D. F. Bray ◽  
E. B. Wagenaar
Keyword(s):  
Potassium Permanganate ◽  
Staining Technique ◽  
Uranyl Acetate ◽  
Staining Procedure ◽  
Lead Citrate ◽  
Double Staining ◽  
Thin Sections ◽  
Citrate Method ◽  
Acetate Lead

A double staining procedure using potassium permanganate and lead citrate is described. The technique, involving treatment of sections with a 1% solution of potassium permanganate followed by lead citrate staining, is shown to be more effective in producing contrast in Spurr-embedded tissue than the conventional uranyl acetate – lead citrate method.

Ultrastructural differences in the exosporium of the Sterne and Vollum strains of Bacillus anthracis

1968 ◽  
Vol 14 (12) ◽  
pp. 1297-1299 ◽  
Author(s):  
M. J. Kramer ◽  
Ivan L. Roth
Keyword(s):  
Electron Microscopy ◽  
Surface Layer ◽  
Bacillus Anthracis ◽  
Potassium Permanganate ◽  
Uranyl Acetate ◽  
Lead Citrate ◽  
Single Application ◽  
Thin Sections ◽  
Acetate Lead ◽  
Different Strains

Spores from three different strains of Bacillus anthracis were examined by electron microscopy for the presence of a hair-like nap previously reported to be present on the exosporium of spores of the Sterne strain (avirulent). In addition to that strain, the Vollum strain (virulent) and a rough, avimlent variant of the Vollum were utilized in the current studies.Spores were fixed with Kellenberger's standard OsO4 fixative and embedded in Maraglas. Thin sections were poststained with various combinations of the following: potassium permanganate, uranyl acetate, lead citrate. The nap on the exosporium of spores of the Sterne strain was revealed most clearly when thin sections were poststained with all of the aforementioned stains. Post-staining by a single application of any of the three reagents resulted in a nap that was barely perceptible.The surface of the exosporium of spores from the Vollum strain and the rough, avirulent variant was found to be quite different from that of the Sterne strain. On the two former, the surface layer is approximately one-third as thick as the layer of hairs in the nap on the latter.

Uranyl Acetate En Bloc Staining to Complement Diagnostic Electron Microscopic Evaluation of Muscle Biopsies

1985 ◽  
Vol 43 ◽  
pp. 462-463
Author(s):  
Caughey R.C. ◽  
Kalyan-Raman U.P.
Keyword(s):  
Plasma Membranes ◽  
Uranyl Acetate ◽  
Staining Procedure ◽  
Lead Citrate ◽  
Ethanol Dehydration ◽  
Electron Microscopic ◽  
En Bloc ◽  
Microscopic Evaluation ◽  
Acetate Lead ◽  
Muscle Biopsies

Uranyl acetate en bloc staining is known to improve overall contrast and membrane preservation and removes glycogen from tissues. This staining procedure complements diagnostic electron microscopic evaluation by enhancing ultrastructural details. The ultrastructural structures enhanced are basement and plasma membranes, mitochondrial membrane and cristae, sarcoplasmic reticulum and T-tubule system. We wanted to study the usefulness of this technique over the conventional method in the study of muscle biopsy. Ultrastructurally, thirty muscle biopsies submitted to our Electron Microscopic Lab were routinely fixed in 2.5% glutaraldehyde and 1% 0S04, dehydrated in a graded series of ethanols, and embedded in Epon 812. Half of the blocks were processed and evaluated with post uranyl acetate/lead citrate staining. The other half were processed and evaluated by en bloc 0. 5% uranyl acetate/Walpole's buffer after 0S04 and before ethanol dehydration. The en bloc half were also stained with post uranyl acetate/lead citrate.

Morphometric comparison of uranyl acetate/lead citrate-stained and peroxidase-stained neutrophil granules

1990 ◽  
Vol 48 (3) ◽  
pp. 756-757
Author(s):  
Charles S. Gilbert ◽  
Richard T. Parmley ◽  
Joseph M. Kinkade
Keyword(s):  
Human Peripheral Blood ◽  
Uranyl Acetate ◽  
Buffy Coat ◽  
Significant Heterogeneity ◽  
Lead Citrate ◽  
Thin Sections ◽  
Low Viscosity ◽  
Physical Density ◽  
Acetate Lead ◽  
Blood Neutrophils

Although neutrophil granules can be broadly divided into peroxidase-positive and peroxidasenegative granules, recent studies have demonstrated significant heterogeneity in neutrophil granule morphology and physical density. The present study was undertaken to evaluate this heterogeneity morphometrically. Human peripheral blood neutrophils were collected from normal volunteers in heparin or EDTA and fixed in 3% glutaraldehyde as a cell suspension or minced buffy coat. An aliquot was stained for myeloperoxidase using 3,3’-diaminobenzidine (DAB) as substrate. All samples were post fixed in 1% OSO4 and embedded in Spurr low viscosity medium. Additionally neutrophil granules isolated according to the method of Rice, et al. were similarly embedded. Thin sections of morphologic preparations were counterstained with methanolic uranyl acetate and aqueous lead citrate (UALC) (Fig. 1), whereas DAB stained specimens were counterstained with LC only (Fig. 2). Attempts to combine UALC and DAB staining in the same section obscured the density imparted by DAB. LC failed to impart significant density to DAB-negative granules in DAB-LC stained specimens resulting in an underestimation of this granule population.

On the Location of Stain in Mitochondria

1968 ◽  
Vol 26 ◽  
pp. 128-129
Author(s):  
G. B. Haydon ◽  
S. O. Smith
Keyword(s):  
Skeletal Muscle ◽  
Heavy Metal ◽  
Oxidative Phosphorylation ◽  
Focal Plane ◽  
Uranyl Acetate ◽  
Thick Section ◽  
Lead Citrate ◽  
Thin Sections ◽  
Thin Sectioning ◽  
Acetate Lead

The degree of matrical staining in isolated and tissue mitochondria has been shown to be a function of their state of oxidative-phosphorylation at the time of fixation (J. Histochem. Cytochem. 15:752, 1967). High resolution EM in conjunction with these studies revealed amplitude contrast images 10 AU and larger in diameter which are independent of the focal plane. These amplitude densities have been interpreted as individual aggregates of heavy metal stain (J. Cell Biol. 35:55A, 1967). Two approaches have been used in an effort to locate such stain aggregates.(1) Four micron epoxy sections of skeletal muscle, cardiac muscle, and liver were stained with uranyl acetate, lead citrate, or both, following the methods used to stain 500 AU sections for EM. After staining, the thick epon sections were re-embedded and thin sections cut perpendicular to the stained surface. Either or both of these metal stains penetrate 500 to 1000 AU into the thick section and give the tissue increased contrast similar to that due to staining after thin sectioning. The mitochondrial matrices are lightly stained indicating that they are not in a state of active oxidative-phosphorylation. Figure 1 shows such mitochondria at low magnification before (Figure 1a) and after (Figure lb) restaining with uranyl acetate and lead citrate. After restaining it is no longer possible to identify the original stained surface.

scholarly journals Immunostaining of DNA in electron microscopy: an amplification and staining procedure for thin sections as alternative to gold labeling.

1994 ◽  
Vol 42 (5) ◽  
pp. 635-643 ◽  
Author(s):  
B Bohrmann ◽  
E Kellenberger
Keyword(s):  
Freeze Substitution ◽  
High Specificity ◽  
Staining Technique ◽  
Uranyl Acetate ◽  
Staining Procedure ◽  
Igg Antibody ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Specificity And Sensitivity ◽  
Eukaryotic Chromatin

We describe a new electron microscopic on-section staining technique with high specificity and sensitivity for DNA-containing structures. Lowicryl HM20 sections of specimens obtained by cryofixation and freeze-substitution are incubated in a first step with a primary IgM antibody specific for double-stranded DNA. The layer of bound antibodies at the section surface is amplified in a successive step by a secondary IgG antibody. Finally, electron scattering of the antibody layer produced is enhanced by staining with a mixture of uranyl acetate and potassium permanganate. The applicability of the method is exemplified by the detection of shape and distribution of various types of bacterial and eukaryotic chromatin.

Membranous alterations in the cytoplasm and nucleus in a primitive neuroectodermal tumor of the brain

1978 ◽  
Vol 36 (2) ◽  
pp. 628-629
Author(s):  
C. N. Sun ◽  
C. Araoz ◽  
H. J. White
Keyword(s):  
Nuclear Envelope ◽  
Tumor Cells ◽  
Osmium Tetroxide ◽  
Primitive Neuroectodermal Tumor ◽  
Uranyl Acetate ◽  
Neuroectodermal Tumor ◽  
Annulate Lamellae ◽  
Lead Citrate ◽  
Thin Sections ◽  
The Brain

The ultrastructure of a cerebral primitive neuroectodermal tumor has been reported previously. In the present case, we will present some unusual previously unreported membranous structures and alterations in the cytoplasm and nucleus of the tumor cells.Specimens were cut into small pieces about 1 mm3 and immediately fixed in 4% glutaraldehyde in phosphate buffer for two hours, then post-fixed in 1% buffered osmium tetroxide for one hour. After dehydration, tissues were embedded in Epon 812. Thin sections were stained with uranyl acetate and lead citrate.In the cytoplasm of the tumor cells, we found paired cisternae (Fig. 1) and annulate lamellae (Fig. 2) noting that the annulate lamellae were sometimes associated with the outer nuclear envelope (Fig. 3). These membranous structures have been reported in other tumor cells. In our case, mitochondrial to nuclear envelope fusions were often noted (Fig. 4). Although this phenomenon was reported in an oncocytoma, their frequency in the present study is quite striking.

Unusual Intranuclear Structures in Chick Sympathetic Neurons

1967 ◽  
Vol 25 ◽  
pp. 188-189
Author(s):  
E. B. Masurovsky ◽  
H. H. Benitez ◽  
M. R. Murray
Keyword(s):  
Electron Microscope ◽  
Sympathetic Neurons ◽  
Uranyl Acetate ◽  
Sympathetic Ganglia ◽  
Lead Citrate ◽  
Thin Sections ◽  
Cns Neurons ◽  
Mammalian Cns

Recent light- and electron microscope studies concerned with the effects of D2O on the development of chick sympathetic ganglia in long-term, organized culture revealed the presence of rod-like fibrillar formations, and associated granulofibrillar bodies, in the nuclei of control and deuterated neurons. Similar fibrillar formations have been reported in the nuclei of certain mammalian CNS neurons; however, related granulofibrillar bodies have not been previously described. Both kinds of intranuclear structures are observed in cultures fixed either in veronal acetate-buffered 2%OsO4 (pH 7. 4), or in 3.5% glutaraldehyde followed by post-osmication. Thin sections from such Epon-embedded cultures were stained with ethanolic uranyl acetate and basic lead citrate for viewing in the electron microscope.

Fine Structure of Interdental Cells in the Mouse Cochlea

1970 ◽  
Vol 28 ◽  
pp. 140-141
Author(s):  
Roberta M. Bruck
Keyword(s):  
Fine Structure ◽  
Electron Microscope ◽  
Young Adult ◽  
Uranyl Acetate ◽  
Ground Substance ◽  
Tectorial Membrane ◽  
Lead Citrate ◽  
Unusual Structure ◽  
Thin Sections ◽  
Collagenous Fibers

An unusual structure in the cochlea is the spiral limbus; this periosteal tissue consists of stellate fibroblasts and collagenous fibers embedded in a translucent ground substance. The collagenous fibers are arranged in vertical columns (the auditory teeth of Haschke). Between the auditory teeth are interdental furrows in which the interdental cells are situated. These epithelial cells supposedly secrete the tectorial membrane.The fine structure of interdental cells in the rat was reported by Iurato (1962). Since the mouse appears to be different, a description of the fine structure of mouse interdental cells' is presented. Young adult C57BL/6J mice were perfused intervascularly with 1% paraformaldehyde/ 1.25% glutaraldehyde in .1M phosphate buffer (pH7.2-7.4). Intact cochlea were decalcified in .1M EDTA by the method of Baird (1967), postosmicated, dehydrated, and embedded in Araldite. Thin sections stained with uranyl acetate and lead citrate were examined in a Phillips EM-200 electron microscope.

Where is the prolamine located in rice endosperm?

1990 ◽  
Vol 48 (3) ◽  
pp. 696-697
Author(s):  
Hsin-Kan Wu ◽  
Mei-Chu Chung
Keyword(s):  
Storage Protein ◽  
Sodium Phosphate ◽  
Recent Experiment ◽  
Protein A ◽  
Uranyl Acetate ◽  
Protein Bodies ◽  
Lead Citrate ◽  
Thin Sections ◽  
Rice Endosperm ◽  
Ethanol Series

In one of our earlier papers (Wu et al. 1978), we suggested that glutelin is the major composition of the round storage protein bodies although they also contain relatively more prolamine than the angular one does. Immunochemical studies of Krishnan et al. (1986) later showed the presence of glutelin in the irregularly-shaped (angular) protein bodies while the prolamines were found in the round ones. Our recent experiment using protein A-gold technique found that prolamine is mainly deposited into the angular protein bodies.Small blocks (1 mm3) of 7 DAF (days after flowering) caryopsis of Orvza perennis were fixed with 3% paraformaldehyde and 3% glutaraldehyde in 0.1M sodium phosphate, pH7.4, dehydrated in a graded ethanol series and infiltrated with Spurr’s resin. Thin sections, after gold labeling, were stained with uranyl acetate and lead citrate. Rabbit antibodies were raised against purified prolamine. Protein A-gold sol complex was prepared based on the technique of Horisberger et al. (1977).

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