Cytochemical staining of connective tissue extracellular matrix with ruthenium hexammine trichloride (RHT)

1990 ◽  
Vol 48 (3) ◽  
pp. 920-921
Author(s):  
Douglas F. Bray
Keyword(s):  
Extracellular Matrix ◽  
Monoclonal Antibodies ◽  
Connective Tissue ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Ruthenium Red ◽  
Fibrous Connective Tissue ◽  
Lead Citrate ◽  
Cytochemical Staining ◽  
Protein Components

The extracellular matrix (ECM) of fibrous connective tissue is a composite of protein- and carbohydrate-containing structures. Preservation of the protein components is achieved through fixation with glutaraldehyde and osmium tetroxide. Carbohydrates however are inadequately preserved and often lost using these fixatives. Cationic dyes such as ruthenium red, alcian blue, and several others have been used to stabilize and retain carbohydrates, particularly the proteoglycans (PGs), but usually with less than optimal results. The present study documents the improved preservation and staining of PGs and other carbohydratecontaining components of skin ECM using ruthenium hexammine trichloride (RHT).Human neonatal foreskins were cut into 1 mm 3 blocks, fixed for 3 h in cacodylate buffered (0.1M, pH 7.2) 2.5% glutaraldehyde containing 1 mg/ml RHT (Polysciences, U.S.A.), postfixed 1 h in buffered 1% OsO4 also containing 1 mg/ml RHT, dehydrated in ethanol and embedded in Spurrs. In some cases the tissue was digested with chondroitinase ABC (Miles, U.S.A.) prior to fixation. Sections from tissue fixed without RHT were poststained in uranyl acetate and lead citrate. Figs. 2 - 5 are from 0.5 μm sections of tissue immunolabelled with monoclonal antibodies to type VI collagen.

Banded Structures in the Connective Tissue of Meningioma

1976 ◽  
Vol 34 ◽  
pp. 234-235
Author(s):  
C. N. Sun ◽  
H. J. White
Keyword(s):  
Connective Tissue ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Collagen Fibrils ◽  
Lead Citrate ◽  
Connective Tissues ◽  
Native Collagen ◽  
Routine Procedures

Previously, we have reported on extracellular cross-striated banded structures in human connective tissues of a variety of organs (1). Since then, more material has been examined and other techniques applied. Recently, we studied a fibrocytic meningioma of the falx. After the specimen was fixed in 4% buffered glutaraldehyde and post-fixed in 1% buffered osmium tetroxide, other routine procedures were followed for embedding in Epon 812. Sections were stained with uranyl acetate and lead citrate. There were numerous cross striated banded structures in aggregated bundle forms found in the connecfive tissue of the tumor. The banded material has a periodicity of about 450 Å and where it assumes a filamentous arrangement, appears to be about 800 Å in diameter. In comparison with the vicinal native collagen fibrils, the banded material Is sometimes about twice the diameter of native collagen.

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.

The Identification of Phospholipid Micelles in Glutaraldehyde-Urea Embedded Tissue

1975 ◽  
Vol 33 ◽  
pp. 494-495
Author(s):  
Daniel C. Pease
Keyword(s):  
Electron Micrographs ◽  
Lung Tissue ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Type Ii ◽  
Lead Citrate ◽  
Phospholipid Micelles ◽  
Type Ii Pneumocytes ◽  
Ultrathin Sections ◽  
Polar Water

It is reasonable to think that phospholipid micelles should be visible and identifiable in electron micrographs of ultrathin sections if only they can be preserved throughout the embedding process. The development of highly polar, water-containing, aminoplastic embedments has made this a likely possibility. With this in mind, an investigation of the lecithin-secreting, Type II pneumocytes of the lung is underway.Initially it has been easiest to recognize phospholipid micelles in lung tissue fixed first with glutaraldehyde, and then secondarily exposed to osmium tetroxide. However, the latter is not a necessary concomitant for micellar preservation. Conventional uranyl acetate and lead citrate staining is finally applied. Importantly, though, the micelles have been most easily seen in tissue embedded in 507. glutaraldehyde polymerized with urea, as described in detail by D.C. Pease and R.G. Peterson (J. Ultra- struct. Res., 41, 133, 1972). When oriented appropriately, the micellar units are seen as tiny, bilayer plates.

scholarly journals Ultrastructural pathology of bioprosthetic heart valves with infectious endocarditis

2021 ◽  
Vol 6 (3) ◽  
pp. 25-34
Author(s):  
R. A. Mukhamadiyarov ◽  
I. V. Milto ◽  
A. G. Kutikhin
Keyword(s):  
Extracellular Matrix ◽  
Heart Valves ◽  
Osmium Tetroxide ◽  
Giant Cells ◽  
Uranyl Acetate ◽  
Diglycidyl Ether ◽  
Multinucleated Giant Cells ◽  
Bioprosthetic Heart Valves ◽  
Sputter Coating ◽  
Backscattered Scanning Electron Microscopy

Aim. To study the ultrastructure of mitral bioprosthetic heart valves (BHVs) which failed due to infective endocarditis.Materials and Methods. Here we examined 7 ethylene glycol diglycidyl ether-treated xenopericardial BHVs excised during repeated BHV replacement because of prosthetic endocarditis. After being fixed in formalin and postfixed in osmium tetroxide, BHVs were dehydrated and stained in uranyl acetate with the subsequent embedding into epoxy resin, grinding, polishing, and lead citrate counterstaining. Upon the sputter coating with carbon, we visualised the BHV microanatomy by means of backscattered scanning electron microscopy at 15 kV voltage.Results. The extracellular matrix underwent degradation and disintegration resulting in loosening, fragmentation, and reduction in the electron density of collagen and elastin fibers. We observed a number of recipient cells (macrophages, multinucleated giant cells, neutrophils, endothelial cells and smooth muscle cells) within the BHVs. The highest number of cells was localized on the valve surfaces. The localization of the recipient cells on the ventricular and atrial surfaces was different. The central part of the valves was abundantly populated by macrophages.Conclusion. Prosthetic endocarditis is accompanied by the migration of recipient cells into the BHV structure, which is the consequence of surface and extracellular matrix disintegration.

An Electron Microscopic Investigation of the Pathogenesis of Hemorrhage Induced by Rattlesnake Venom

1974 ◽  
Vol 32 ◽  
pp. 56-57
Author(s):  
Charlotte L. Ownby ◽  
Robert A. Kainer ◽  
Anthony T. Tu
Keyword(s):  
Phosphate Buffer ◽  
Osmium Tetroxide ◽  
Microscopic Investigation ◽  
Uranyl Acetate ◽  
Electron Microscopic Investigation ◽  
Lead Citrate ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Rattlesnake Venom ◽  
Diamondback Rattlesnake

One of the significant changes induced by the injection of rattlesnake (Crotalidae) venom is hemorrhage. Since crotaline antivenin does not prevent such local tissue damage, a more effective treatment of snakebite is needed. To aid in the development of such a treatment the pathogenesis of venom-induced hemorrhae was investigated.Swiss-Webster white mice were injected intramuscularly with Western diamondback rattlesnake (Crotalus atrox) venom. Two minutes after the injection, muscle tissue was obtained by bioosy from the thigh and fixed in 6% glutaraldehyde in Milloniq's phosphate buffer (DH 7.4, 2 hrs., 4°C). After post-fixation in 2% osmium tetroxide in Milloniq's phosphate buffer (pH 7.4, 1hr., 4°C) the tissue was dehydrated routinely in ethanol and embedded in Epon 812. The thin sections were stained with uranyl acetate in methanol and lead citrate then observed with either a Zeiss EM 9A or an Hitachi HS-8 electron microscope.

Ultrastructural Morphology of the Anagen Stage Hair Follicle of Male Beagle Dogs

1977 ◽  
Vol 35 ◽  
pp. 652-653
Author(s):  
F. A. Al-Bagdadi ◽  
C. W. Titkemeyer ◽  
J. E. Lovell
Keyword(s):  
Electron Microscope ◽  
Basement Membrane ◽  
Skin Biopsy ◽  
Osmium Tetroxide ◽  
Dermal Papilla ◽  
Uranyl Acetate ◽  
Ground Substance ◽  
Lead Citrate ◽  
Beagle Dogs ◽  
Cytoplasmic Processes

Skin biopsy samples were collected monthly from the lateral sides of 9 male Beagle dogs over a period of 1 year. The samples were fixed in 3% gluteral- dehyde and post fixed in 1% osmium tetroxide using phosphate or S-Collidine as buffers. They were dehydrated, embedded in Epon 812, sectioned with an LKB Ultrotome III, and stained with Reynolds' lead citrate and 1% uranyl acetate. They were examined and photographed by use of an RCA-3H electron microscope.The dermal papilla during anagen consisted of fibroblast-like cells some of which had cytoplasmic processes. The cytoplasm contained many mitochondria. (Fig. 1) The cells of the dermal papilla were either peripherally arranged spindle-shaped cells or polygonal cells. The basement membrane was either related to the fibroblast cytoplasmic processes or was free of the cytoplasmic processes but with a thick zone composed of fibers, granular ground substance and spaces.

Sebaceous Basal Cell Carcinoma - An Ultrastructural Study

1982 ◽  
Vol 40 ◽  
pp. 232-233
Author(s):  
Annette M. Andrews ◽  
Boon-Nam Blackwell ◽  
Terrell R. Hoage ◽  
Fred F. Kadiubar
Keyword(s):  
Basal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Basal Cell ◽  
Sebaceous Gland ◽  
Osmium Tetroxide ◽  
Wistar Rat ◽  
Uranyl Acetate ◽  
Lead Citrate ◽  
Tumor Specimen ◽  
Standard Procedures

Basal cell carcinoma with sebaceous gland differentiation has been described in mice and humans but spontaneous skin tumors of this type are uncommon in the rat.The tumor specimen was taken from a 26 month old male Wistar rat. Electron microscopy specimens were fixed by immersion in cacodylatebuffered 4% glutaraldehyde, post-fixed in 1% osmium tetroxide, dehydrated, cleared and embedded by standard procedures. Semi-thin (μm) sections were stained with ethanolic uranyl acetate followed by lead citrate, then examined on a Philips EM201 electron microscope.

Electron Microscopy of Wound Healing in Patients with Hernia

1975 ◽  
Vol 33 ◽  
pp. 352-353
Author(s):  
C.N. Sun ◽  
H.J. White ◽  
R.C. Read
Keyword(s):  
Electron Microscopy ◽  
Wound Healing ◽  
Phosphate Buffer ◽  
Osmium Tetroxide ◽  
Phosphotungstic Acid ◽  
Rectus Sheath ◽  
Uranyl Acetate ◽  
Collagen Fibrils ◽  
Lead Citrate ◽  
Native Collagen

Previously we have reported the defect of collagen fibrils from herniated rectus sheath. This presentation includes additional sections from postsurgical incisions (10 days) from both control and hernia patients. Small pieces of rectus sheath were fixed in 3% glutaraldehyde in phosphate buffer (pH 7.2) and post fixed with buffered 2% osmium tetroxide. The tissues were then dehydrated in serially increasing concentrations of alcohol and embedded in Epon 812. Sections were stained with 2.5% phosphotungstic acid or uranyl acetate and lead citrate.Previously we found that collagen fibrils from "non-herniated" rectus sheath have uniform diameters and 640 Å periodicity with seven or more intraperiodic bands resembling typical native collagen fibrils, while the fibrils from fascia obtained from patients with direct herniation show considerable variation in diameter. These variations are often found in the same individual fibers with a range from 300 Å to 3000 Å.

A Modified Technic of Staining Elastic Tissue for Electron Microscopic Study

1975 ◽  
Vol 33 ◽  
pp. 626-627
Author(s):  
J.A. Nordquist ◽  
K. Chrysant ◽  
A.K. Mandal
Keyword(s):  
Electron Microscopy ◽  
Osmium Tetroxide ◽  
Renal Tissue ◽  
Uranyl Acetate ◽  
Elastic Tissue ◽  
Lead Citrate ◽  
Electron Microscopic ◽  
Modified Method ◽  
Tetraphenyl Porphyrin ◽  
Normal Rat

By electron microscopy elastic tissue appear electrolucent in osmium fixed unstained grids as well as grids stained with uranyl acetate and lead citrate (UA + LC). Albert and Fleischer have studied aorta of mice with metalloporphyrins imparting conspicuous electron density to the elastic tissue. We are reporting here a modified method of electron microscopic (EM) study of the elastic tissue using metalloporhyrin, silver tetraphenyl porphyrin sulfonate (STPPS).We have studied the renal arterioles of rats and human in normal and diseased states. Elastic tissue of the aorta from young normal rat served as control for this study. Renal and aortic tissues were fixed in 4 percent glutaraldehyde, post fixed in 1 percent osmium tetroxide and embedded in spurr (blocks). From the blocks of renal tissue, 0.5 μ sections were cut, stained with methylene blue and azure II and studied by light microscopy.

scholarly journals Electron microscopic immunoperoxidase staining of insulin using 4-chloro-1-naphthol after osmium fixation.

1982 ◽  
Vol 30 (7) ◽  
pp. 710-712 ◽  
Author(s):  
D G Baskin ◽  
H Mar ◽  
K C Gorray ◽  
W Y Fujimoto
Keyword(s):  
Osmium Tetroxide ◽  
Ultrastructural Localization ◽  
Uranyl Acetate ◽  
Immunoperoxidase Staining ◽  
Lead Citrate ◽  
Electron Microscopic ◽  
Specific Staining ◽  
Fixed Tissue ◽  
Substrate Solution ◽  
Peptide Antigens

Ultrastructural localization of insulin in B cells of guinea pig pancreas was accomplished after osmium fixation with an immunoperoxidase procedure that utilized 4-chloro-1-naphthol (CN) in the substrate solution. The principal features of this protocol were: a) osmium tetroxide postfixation; b) omission of hydrogen peroxide "etching"; c) use of CN instead of diaminobenzidine in the substrate solution; d) elimination of osmium tetroxide after the substrate reaction; e) uranyl acetate and lead citrate counterstaining. This procedure produces intense specific staining with low background using highly dilute antiserum, and appears to be useful for postembedding immunoperoxidase staining of a variety of peptide antigens in osmium-fixed tissue.

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