A rapid dual staining procedure for the quantitative discrimination of prion amyloid from tissues reveals how interactions between amyloid and lipids in tissue homogenates may hinder the detection of prions

2009 ◽  
Vol 77 (1) ◽  
pp. 90-97 ◽  
Author(s):  
R. Hervé ◽  
R. Collin ◽  
H.E. Pinchin ◽  
T. Secker ◽  
C.W. Keevil
Keyword(s):  
Staining Procedure ◽  
Tissue Homogenates ◽  
Dual Staining

scholarly journals Discrimination between Viable and Dead Encephalitozoon cuniculi (Microsporidian) Spores by Dual Staining with Sytox Green and Calcofluor White M2R

2000 ◽  
Vol 38 (10) ◽  
pp. 3811-3814 ◽  
Author(s):  
L. C. Green ◽  
P. J. LeBlanc ◽  
E. S. Didier
Keyword(s):  
Systemic Disease ◽  
Organ Transplant ◽  
Spore Wall ◽  
Staining Procedure ◽  
Excitation Wavelength ◽  
Encephalitozoon Cuniculi ◽  
Calcofluor White ◽  
Sytox Green ◽  
Intracellular Parasites ◽  
Dual Staining

Microsporidia are obligate intracellular parasites, recognized as causing chronic diarrhea and systemic disease in AIDS patients, organ transplant recipients, travelers, and malnourished children. Species of microsporidia that infect humans have been detected in drinking-water sources, and methods are needed to ascertain if these microsporidia are viable and capable of causing infections. In this study, Calcofluor White M2R and Sytox Green stains were used in combination to differentiate between live (freshly harvested) and dead (boiled)Encephalitozoon cuniculi spores. Calcofluor White M2R binds to chitin in the microsporidian spore wall. Dual-stained live spores appeared as turquoise-blue ovals, while dead spores appeared as white-yellow ovals at an excitation wavelength of 395 to 415 nm used for viewing the Calcofluor stain. Sytox Green, a nuclear stain, is excluded by live spores but penetrates compromised spore membranes. Dual-stained dead spores fluoresced bright yellow-green when viewed at an excitation wavelength of 470 to 490 nm, whereas live spores failed to stain with Sytox Green. After live and dead spores were mixed at various ratios, the number of viably stained spores detected in the dual-staining procedure correlated (P = 0.0025) with the expected numbers of viable spores. Spore mixtures were also assayed for infectivity in a focus-forming assay, and a correlation (P = 0.0002) was measured between the percentage of focus-forming microsporidia and the percentage of expected infectious spores in each mixture. By analysis of variance, no statistically significant differences were measured between the percentage of viably stained microsporidia and the percentage of infectious microsporidia (P = 0.964) in each mixture. These results suggest that Calcofluor White M2R and Sytox Green stains, when used together, may facilitate studies to identify viable microsporidia.

scholarly journals Evaluation of a Dual-Staining Method for Acid-Fast Bacilli

1975 ◽  
Vol 2 (2) ◽  
pp. 149-150
Author(s):  
N. M. Burdash ◽  
M. E. West ◽  
E. R. Bannister ◽  
C. Dyar ◽  
R. C. Duncan
Keyword(s):  
Staining Method ◽  
Staining Procedure ◽  
Poor Correlation ◽  
Acid Fast Bacilli ◽  
Staining Techniques ◽  
Dual Staining

A dual-staining procedure for acid-fast bacilli was found to have poor correlation with the Ziehl-Neelsen and auramine-rhodamine staining techniques.

Estrogen and progesterone receptors in carcinoma of the breast (A cytochemical, immunofluorescence and electron microscopic study)

1986 ◽  
Vol 44 ◽  
pp. 116-117
Author(s):  
Robert L. Elliott ◽  
Gregory O. Harrison ◽  
Anne Coble-Landry ◽  
Lindsay Bullock-Ledford ◽  
Anne T. McRea ◽  
...  
Keyword(s):  
Ductal Carcinoma ◽  
Progesterone Receptors ◽  
Uranyl Acetate ◽  
Staining Procedure ◽  
Electron Microscopic ◽  
Carcinoma Of The Breast ◽  
Thin Sections ◽  
Infiltrating Ductal Carcinoma ◽  
Estrogen And Progesterone Receptor ◽  
Dual Staining

Tissue from 105 patients with infiltrating ductal carcinoma of the breast was assayed for the estrogen and progesterone receptor by the cytochemical dextran coated charcoal technique and by immunofluorescence with a direct dual staining procedure with Fluorocep, and also examined electron microscopically.Tissue was fixed in Zambonis; post-fixed in 1% OsO4; embedded in a Med Cast Resin, and thin sections were cut on a ultramicrotone. They were stained with uranyl acetate and lead citrate and examined in a Hitachi H-600 electron microscope.

Ca-Histochemistry in slime mold plasmodia

1978 ◽  
Vol 36 (2) ◽  
pp. 130-131
Author(s):  
Ulrich Dierkes
Keyword(s):  
Physarum Polycephalum ◽  
Carbon Coating ◽  
Freeze Drying ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Slime Mold ◽  
Staining Procedure ◽  
Protoplasmic Streaming ◽  
Intracellular Ca

Calcium is supposed to play an important role in the control of protoplasmic streaming in slime mold plasmodia. The motive force for protoplasmic streaming is generated by the interaction of actin and myosin. This contraction is supposed to be controlled by intracellular Ca-fluxes similar to the triggering system in skeleton muscle. The histochemical localisation of calcium however is problematic because of the possible diffusion artifacts especially in aquous media.To evaluate this problem calcium localisation was studied in small pieces of shock frozen (liquid propane at -189°C) plasmodial strands of Physarum polycephalum, which were further processed with 3 different methods: 1) freeze substitution in ethanol at -75°C, staining in 100% ethanol with 1% uranyl acetate, and embedding in styrene-methacrylate. For comparison the staining procedure was omitted in some preparations. 2)Freeze drying at about -95°C, followed by immersion with 100% ethanol containing 1% uranyl acetate, and embedding. 3) Freeze fracture, carbon coating and SEM investigation at temperatures below -100° C.

En face dual fluorescence staining of fatty streaks allows observation of initiation and progression of atherosclerotic lesions

1995 ◽  
Vol 53 ◽  
pp. 864-865
Author(s):  
Anne M. Klinkner ◽  
Crystal R. Waites ◽  
Peter J. Bugelski ◽  
William D. Kerns
Keyword(s):  
Fluorescent Dyes ◽  
Nile Red ◽  
Dimethyl Formamide ◽  
High Cholesterol Diet ◽  
Methods Development ◽  
Atherosclerotic Lesions ◽  
En Face ◽  
Biochemical Evaluation ◽  
Stock Solutions ◽  
Dual Staining

A primary effort in the understanding of the progression of atherosclerotic disease has been methods development for visualization of the atherosclerotic plaque. We introduce a new method for the qualitative analysis of lipids in atherosclerotic fatty streaks which also retains those lipids for biochemical evaluation. An original aspect of the process is the ability to view an entire fatty streak en face, selectively stained for specific lipid classes within the lesion.New Zealand white rabbits were fed a high cholesterol diet(0.15%-0.3% for 14 wks). The aorta was removed and fixed in Carson's phosphate buffered formaldehyde followed by dual staining in the fluorescent dyes Nile red and filipin. Stock solutions of nile red(0.5mg/ml acetone) and filipin(2.5mg/ml dimethyl formamide) were prepared and kept at -20°C; all subsequent steps were at RT. 0.5cm × 1.0cm pieces of aorta were trimmed and adventitia removed. The pieces were then washed 3×15 min in PBS w/o CaMg, soaked in Nile red(NR)/filipin(Fl) stain(100(il NR stock + 200μl Fl stock in 10 ml PBS for 30 min, washed in PBS 3×30 min, rinsed with distilled water, mounted(Crystal Mount, Biomedia) and coverslipped and viewed by fluorescence microscopy.

A pre-embedding double staining procedure used to observe the effect of fixation on the activity of intercellular adhesion molecule on T and B cells

1990 ◽  
Vol 48 (3) ◽  
pp. 378-379
Author(s):  
Jane E. Ramberg ◽  
Shigeto Tohma ◽  
Peter E. Lipsky
Keyword(s):  
B Cells ◽  
Adhesion Molecule ◽  
Colloidal Gold ◽  
Intercellular Adhesion ◽  
Intercellular Adhesion Molecule ◽  
Staining Procedure ◽  
Double Staining ◽  
T And B Cells ◽  
Microtiter Plates ◽  
Streptavidin Peroxidase

Intercellular adhesion molecule (ICAM-1) appears to be a ligand for LFA-1 dependent adhesion in T cell mediated cytotoxcity. It is found on cells of both hematopoietic and non-hematopoietic origin. While observing the activity of ICAM-1 on the surfaces of interacting T and B cells, we found that we could successfully carry out a pre-embedding double staining procedure utilizing both colloidal gold and peroxidase conjugated reagents.On 24-well microtiter plates, mitomycin-treated T4 cells were stimulated with 64.1 (anti-CD3) for one hour before the addition, in some instances, of B cells. Following a 12-48 hour incubation at 38°C, the cells were washed and then immunostained with a colloidal gold conjugated RFB-4 (anti-CD22); biotinylated R6.5 (anti-ICAM-1); followed by streptavidin/peroxidase. This method allowed us to observe two different antigens without concern about possible cross-reaction of reagents. Because we suspected ICAM-1 and R6.5 were sensitive to fixation, we tried varying concentrations of fresh paraformaldehyde before R6.5, after R6.5 and after streptavidin/peroxidase. All immunostaining and washing was done on ice with ice cold reagents.

Solvent-assisted osmium staining of butyl acrylate and ethylene-propylene-diene in a styrene acrylonitrile matrix

1993 ◽  
Vol 51 ◽  
pp. 900-901 ◽  
Author(s):  
Gudrun A. Hutchins
Keyword(s):  
Butyl Acrylate ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Staining Procedure ◽  
Reactive Site ◽  
Ethylene Propylene ◽  
Minimal Distortion ◽  
Styrene Acrylonitrile ◽  
Engineering Polymers ◽  
Effective Reaction

In order to optimize the toughening effect of elastomers in engineering polymers, it is necessary to characterize the size, morphology and dispersion of the specific elastomer within the polymer matrix. For unsaturated elastomers such as butadiene or isoprene, staining with osmium tetroxide is a well established procedure. The residual carbon-carbon double bond in these materials is the reactive site and forms a 1,2-dilato complex with the OsO4. Incorporation of osmium tetroxide into the elastomer not only produces sufficient contrast for TEM, but also crosslinks the elastomer sufficiently so that ultramicrotomy can be accomplished at room temperature with minimal distortion.Blends containing saturated elastomers such as butyl acrylate (BA) and ethylene propylene diene monomer (EPDM) cannot be stained directly with OsO4 because effective reaction sites such as C=C or -NH2 are not available in sufficient number. If additional reaction sites can be introduced selectively into the elastomer by a chemical reaction or the absorption of a solvent, a modified, two-step osmium staining procedure is possible.

Isolation and characterization of cell walls from the mesocarp of mature grape berries (Vitis vinifera)

2020 ◽  
Author(s):  
KJ Nunan ◽  
Ian Sims ◽  
A Bacic ◽  
SP Robinson ◽  
GB Fincher
Keyword(s):  
Vitis Vinifera ◽  
Cell Walls ◽  
Vascular Tissue ◽  
Compositional Analysis ◽  
Water Soluble ◽  
Vitis Vinifera L ◽  
Nylon Mesh ◽  
Cytoplasmic Material ◽  
Isolation And Characterization ◽  
Tissue Homogenates

Cell walls have been isolated from the mesocarp of mature grape (Vitis vinifera L.) berries. Tissue homogenates were suspended in 80% (v/v) ethanol to minimise the loss of water-soluble wall components and wet-sieved on nylon mesh to remove cytoplasmic material. The cell wall fragments retained on the sieve were subsequently treated with buffered phenol at pH 7.0, to inactivate any wall-bound enzymes and to dislodge small amounts of cytoplasmic proteins that adhered to the walls. Finally, the wall preparation was washed with chloroform/methanol (1:1, v/v) to remove lipids and dried by solvent exchange. Scanning electron microscopy showed that the wall preparation was essentially free of vascular tissue and adventitious protein of cytoplasmic origin. Compositional analysis showed that the walls consisted of approximately 90% by weight of polysaccharide and less than 10% protein. The protein component of the walls was shown to be rich in arginine and hydroxyproline residues. Cellulose and polygalacturonans were the major constituents, and each accounted for 30-40% by weight of the polysaccharide component of the walls. Substantial varietal differences were observed in the relative abundance of these two polysaccharides. Xyloglucans constituted approximately 10% of the polysaccharide fraction and the remainder was made up of smaller amounts of mannans, heteroxylans, arabinans and galactans.

THE CONTRIBUTION OF LACTATE AND PHOSPHATE ANIONS TO THE BUFFER PROPERTIES OF TISSUE HOMOGENATES OF BREAST ADENOCARCINOMA

2019 ◽  
Vol 65 (5) ◽  
pp. 684-690
Author(s):  
Margarita Barsukova ◽  
Yekaterina Khomutova ◽  
Yevgeniy Khomutov
Keyword(s):  
Mammary Gland ◽  
Buffer Capacity ◽  
Tumor Tissue ◽  
Mammary Adenocarcinoma ◽  
Breast Adenocarcinoma ◽  
Acid Base ◽  
Adjacent Tissue ◽  
Tumor Tissues ◽  
Tissue Homogenates ◽  
Phosphate Anions

The article discusses the role of conjugated lactic acid/ lactate anion (LacH/Lac-) and dihydrogenphosphate anion/ hydrogenphosphate anion (H2PO4-/HPO42-) pairs in the formation of the buffer properties of tissue as a factor determining pH. The buffer properties of homogenates of the tissue of adenocarcinoma of the mammary gland and the adjacent tissue were quantitatively characterized by the buffer capacity which was determined by potentiometric titration. The concentrations of acid anions were determined spectrophotometrically. The material was biopsy specimens of mammary gland adenocarcinoma (T1-4, N0-1, M0) and adjacent tissue of 22 patients aged from 33 to 75 years. It was found that the buffer capacity of tumors is in 2.5 times higher than in normal tissue. It was established that for the tumor tissue, the buffer capacity of the LacH/Lac- system is in 3 times higher, and the buffer capacity of the H2PO4-/HPO42-system is in 2.5 times greater than for normal untransformed tissue. Concentrations of lactate anions (1,93 ± 0,50 vs 0,57 ± 0,22; p <0.001) and phosphate anions (2,54 ± 0,39 vs 0,70 ± 0,19; p <0,001) in homogenates of the tumor tissue were significantly higher in tumor tissue in comparison with the adjacent tissue. A strong correlation was found between the concentration of phosphate anions and the buffer capacity for tumor tissue (r = 0,857; p = 0,002) and for adjacent tissue (r = 0,917; p <0,001). The correlation between the concentration of lactate anions and the buffer capacity for tumor tissues can be estimated as average (r = 0,626; p = 0,053), while it is absent for the adjacent tissue (r = 0,494; p = 0,147). The results suggest that the acid-base properties of homogenates of mammary adenocarcinoma tissues are determined by two buffer systems: LacH/Lac- and H2PO4-/HPO42-, while the intracellular acid-base homeostasis of non-transformed tissues is mainly determined by the H2PO4-/HPO42- system.

scholarly journals Axon typing of rat muscle nerves using a double staining procedure for cholinesterase and carbonic anhydrase.

1991 ◽  
Vol 39 (12) ◽  
pp. 1617-1625 ◽  
Author(s):  
M J Szabolcs ◽  
A Windisch ◽  
R Koller ◽  
M Pensch
Keyword(s):  
Carbonic Anhydrase ◽  
Cross Sections ◽  
Sensory Nerve ◽  
Histological Section ◽  
Trapezius Muscle ◽  
Motor Units ◽  
Conventional Technique ◽  
Nerve Fibers ◽  
Staining Procedure ◽  
Muscle Nerve

We developed a method for detecting activity of axonal cholinesterase (CE) and carbonic anhydrase (CA)--markers for motor and sensory nerve fibers (NFs)--in the same histological section. To reach this goal, cross-sections of muscle nerves were sequentially incubated with the standard protocols for CE and CA histochemistry. A modified incubation medium was used for CA in which Co++ is replaced by Ni++. This avoids interference of the two histochemical reactions because Co++ binds unspecifically to the brown copper-ferroferricyanide complex representing CE activity, whereas Ni++ does not. Cross-sections of the trapezius muscle nerve containing efferent and afferent NFs in segregated fascicles showed that CE activity was confined to motor NFs. Axonal CA was detected solely in sensory NFs. The number of labeled motor and sensory NFs determined in serial cross-sections stained with either the new or the conventional technique was not significantly different. Morphometric analysis revealed that small unreactive NFs (diameter less than 5 microns) are afferent, medium-sized ones (5 microns less than d less than 7 microns) are unclassifiable, and large ones (d greater than 7 microns) are efferent. The heterogenous CE activity of thick (alpha) motor NFs is linked to the type of their motor units. "Fast" motor units contain CE reactive NFs; "slow" ones have CE negative neurites.

Sign in / Sign up

Export Citation Format

Share Document