Histochemical Detection of Collagen Fibers by Sirius Red/Fast Green Is More Sensitive than van Gieson or Sirius Red Alone in Normal and Inflamed Rat Colon

The ribbon-like macronucleus of Euplotes eurystomus pinches in half amitotically at each cell division. Several hours before the actual division two lightly staining duplication bands (reorganization bands) appear at the ends of the nucleus and approach each other slowly, finally meeting near the middle. Distal to the bands, that is, in regions through which the bands have already passed, the concentration of DNA (Feulgen) and "histone" (alkaline fast green) is greater than in the central zone. These facts suggest the hypothesis that DNA-histone synthesis takes place in a sequential fashion starting at the tips of the nucleus and proceeding to the middle. That this hypothesis is correct is shown by autoradiographic and photometric observations. Tritium-labelled thymidine is incorporated only in a limited region immediately distal to the bands. The average amount of Feulgen dye bound by the nucleus rises as the duplication bands approach each other, and is double the presynthesis value by the time the bands meet. A similar rise in the alkaline fast green dye is seen in duplicating nuclei, although no completely post-synthesis values were obtained in this study. The quantitative data are consistent with the assumption that the macronucleus contains a number of DNA-histone "units," presumably chromosomes, each of which duplicates once and only once.

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Proteomics study of the antifibrotic effects of α-mangostin in a rat model of renal fibrosis

Asian Biomedicine ◽

10.1515/abm-2019-0015 ◽

2019 ◽

Vol 12 (4) ◽

pp. 149-160

Author(s):

Thana Chaeyklinthes ◽

Vilailak Tiyao ◽

Sittiruk Roytrakul ◽

Narumon Phaonakrop ◽

Udomsri Showpittapornchai ◽

...

Keyword(s):

Renal Fibrosis ◽

Treated Group ◽

Renal Tissue ◽

Collagen Fibers ◽

Mitogen Activated Protein Kinase ◽

Control Group ◽

Interacting Protein ◽

Amino Terminal ◽

Male Wistar Rats ◽

Sirius Red

AbstractBackgroundRenal fibrosis is a consequence of a “faulty” wound-healing mechanism that results in the accumulation of extracellular matrix, which could lead to the impairment of renal functions. α-Mangostin (AM) may prevent the formation of liver fibrosis, but there has yet to be a conclusive investigation of its effect on renal fibrosis.ObjectivesTo investigate the renoprotective effect of AM against thioacetamide (TAA)-induced renal fibrosis in rats at the morphological and proteomic levels.MethodsWe divided 18 male Wistar rats into 3 groups: a control group, a TAA-treated group, and a TAA + AM group. The various agents used to treat the rats were administered intraperitoneally over 8 weeks. Subsequently, the morphology of renal tissue was analyzed by histology using Sirius Red staining and the relative amount of stained collagen fibers quantified using ImageJ analysis. One-dimensional gel liquid chromatography with tandem mass spectrometry (GeLC-MS/MS) was used to track levels of protein expression. Proteomic bioinformatics tools including STITCH were used to correlate the levels of markers known to be involved in fibrosis with Sirius Red-stained collagen scoring.ResultsHistology revealed that AM could reduce the relative amount of collagen fibers significantly compared with the TAA group. Proteomic analysis revealed the levels of 4 proteins were modulated by AM, namely CASP8 and FADD-like apoptosis regulator (Cflar), Ragulator complex protein LAMTOR3 (Lamtor3), mitogen-activated protein kinase kinase kinase 14 (Map3k14), and C-Jun-amino-terminal kinase-interacting protein 3 (Mapk8ip3).ConclusionAM can attenuate renal fibrosis by the suppression of pathways involving Cflar, Lamtor3, Map3k14, and Mapk8ip3.

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Safranin O without fast green is the best staining method for testing the degradation of macromolecules in a cartilage extracellular matrix for the determination of the postmortem interval

Forensic Science Medicine and Pathology ◽

10.1007/s12024-019-00208-0 ◽

2019 ◽

Vol 16 (2) ◽

pp. 252-258 ◽

Cited By ~ 1

Author(s):

Armin Alibegović ◽

Rok Blagus ◽

Inigo Zubiavrre Martinez

Keyword(s):

Extracellular Matrix ◽

Staining Method ◽

Postmortem Interval ◽

Fast Green ◽

Green Is ◽

Cartilage Extracellular Matrix ◽

Safranin O

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Bacterial-Mucosal Cell Junctional Complexes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100050731 ◽

1974 ◽

Vol 32 ◽

pp. 144-145

Author(s):

Roger C. Wagner

Keyword(s):

Intestinal Wall ◽

Rat Colon ◽

Mucosal Cell ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Mucosal Cells ◽

Mucosal Lining ◽

Degenerative Alterations ◽

Junctional Complexes ◽

Intestinal Mucosal Cells

Bacteria exhibit the ability to adhere to the apical surfaces of intestinal mucosal cells. These attachments either precede invasion of the intestinal wall by the bacteria with accompanying inflammation and degeneration of the mucosa or represent permanent anchoring sites where the bacteria never totally penetrate the mucosal cells.Endemic gram negative bacteria were found attached to the surface of mucosal cells lining the walls of crypts in the rat colon. The bacteria did not intrude deeper than 0.5 urn into the mucosal cells and no degenerative alterations were detectable in the mucosal lining.

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Methionine-Specific Staining of Collagen

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010005408x ◽

1982 ◽

Vol 40 ◽

pp. 294-295

Author(s):

E.M. Kuhn ◽

K.D. Marenus ◽

M. Beer

Keyword(s):

Amino Acids ◽

Collagen Fibers ◽

Type Iii Collagen ◽

Type I ◽

Electron Microscopic ◽

Good Correspondence ◽

Specific Staining ◽

Type Iii ◽

Different Types ◽

Sulfur Containing

Fibers composed of different types of collagen cannot be differentiated by conventional electron microscopic stains. We are developing staining procedures aimed at identifying collagen fibers of different types.Pt(Gly-L-Met)Cl binds specifically to sulfur-containing amino acids. Different collagens have methionine (met) residues at somewhat different positions. A good correspondence has been reported between known met positions and Pt(GLM) bands in rat Type I SLS (collagen aggregates in which molecules lie adjacent to each other in exact register). We have confirmed this relationship in Type III collagen SLS (Fig. 1).

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Selective Staining of Acid Mucopolysaccharides By Ruthenium Red

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100099933 ◽

1968 ◽

Vol 26 ◽

pp. 38-39

Author(s):

J. H. Luft

Keyword(s):

Electron Microscope ◽

Light Microscopy ◽

Light Microscope ◽

Osmium Tetroxide ◽

Single Cells ◽

Ruthenium Red ◽

Collagen Fibers ◽

Selective Staining ◽

Pectic Substances ◽

Solid Tissues

Ruthenium red is one of the few completely inorganic dyes used to stain tissues for light microscopy. This novelty is enhanced by ignorance regarding its staining mechanism. However, its continued usefulness in botany for demonstrating pectic substances attests to selectivity of some sort. Whether understood or not, histochemists continue to be grateful for small favors.Ruthenium red can also be used with the electron microscope. If single cells are exposed to ruthenium red solution, sufficient mass can be bound to produce observable density in the electron microscope. Generally, this effect is not useful with solid tissues because the contrast is wasted on the damaged cells at the block surface, with little dye diffusing more than 25-50 μ into the interior. Although these traces of ruthenium red which penetrate between and around cells are visible in the light microscope, they produce negligible contrast in the electron microscope. However, its presence can be amplified by a reaction with osmium tetroxide, probably catalytically, to be easily visible by EM. Now the density is clearly seen to be extracellular and closely associated with collagen fibers (Fig. 1).

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Ultrastructural Study of Rat Colon Following Psyllium Ingestion

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100119685 ◽

1985 ◽

Vol 43 ◽

pp. 580-581

Author(s):

F.G. Lightfoot ◽

L.E. Grau ◽

M.M. Cassidy ◽

G.R. Tadvalkar ◽

G.V. Vahouny

Keyword(s):

Electron Microscopy ◽

Morphological Changes ◽

Large Population ◽

Rat Colon ◽

Gastrointestinal Disorders ◽

Luminal Surface ◽

Fecal Material ◽

Transmission Electron ◽

Morphologic Analysis ◽

Irritable Bowel

Psyllium hydrophillic mucilloid is a natural gelling fiber consumed by a large population of our society. It is used as a bulk-producing laxative and in the treatment of gastrointestinal disorders such as “Irritable Bowel Syndrome”. The literature pertaining to the ultrastructural effects of this agent is sparse.This study documents morphological changes induced by psyllium. Animals fed a diet containing 2% psyllium for four weeks were subsequently sacrificed and processed for scanning and transmission electron microscopy. The colon contained fecal material combined with psyllium which conformed to the contour of the luminal surface. This mixture formed surface replicas of the intestinal mucosa. These replicas and their related colonic sites were processed for morphologic analysis.

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Biological and biomedical applications of collagenous biomaterials

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100161849 ◽

1990 ◽

Vol 48 (3) ◽

pp. 856-857

Author(s):

Yasushi P. Kato ◽

Michael G. Dunn ◽

Frederick H. Silver ◽

Arthur J. Wasserman

Keyword(s):

Biomedical Applications ◽

Soft Tissues ◽

Collagen Fibers ◽

Bone Collagen ◽

Cover Slip ◽

Fibroblast Migration ◽

Cellular Expression ◽

Defect Repair

Collagenous biomaterials have been used for growing cells in vitro as well as for augmentation and replacement of hard and soft tissues. The substratum used for culturing cells is implicated in the modulation of phenotypic cellular expression, cellular orientation and adhesion. Collagen may have a strong influence on these cellular parameters when used as a substrate in vitro. Clinically, collagen has many applications to wound healing including, skin and bone substitution, tendon, ligament, and nerve replacement. In this report we demonstrate two uses of collagen. First as a fiber to support fibroblast growth in vitro, and second as a demineralized bone/collagen sponge for radial bone defect repair in vivo.For the in vitro study, collagen fibers were prepared as described previously. Primary rat tendon fibroblasts (1° RTF) were isolated and cultured for 5 days on 1 X 15 mm sterile cover slips. Six to seven collagen fibers, were glued parallel to each other onto a circular cover slip (D=18mm) and the 1 X 15mm cover slip populated with 1° RTF was placed at the center perpendicular to the collagen fibers. Fibroblast migration from the 1 x 15mm cover slip onto and along the collagen fibers was measured daily using a phase contrast microscope (Olympus CK-2) with a calibrated eyepiece. Migratory rates for fibroblasts were determined from 36 fibers over 4 days.

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Facilitated migration of cells from a transected peripheral nerve over collagen fibers: in vivo observations

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156420 ◽

1989 ◽

Vol 47 ◽

pp. 888-889

Author(s):

Arthur J. Wasserman ◽

Azam Rizvi ◽

George Zazanis ◽

Frederick H. Silver

Keyword(s):

Sciatic Nerve ◽

Peripheral Nerve ◽

Collagen Gel ◽

Collagen Fibers ◽

Control Group ◽

Guide Tube ◽

Sprague Dawley ◽

Silicone Tube ◽

Thin Sections

In cases of peripheral nerve damage the gap between proximal and distal stumps can be closed by suturing the ends together, using a nerve graft, or by nerve tubulization. Suturing allows regeneration but does not prevent formation of painful neuromas which adhere to adjacent tissues. Autografts are not reported to be as good as tubulization and require a second surgical site with additional risks and complications. Tubulization involves implanting a nerve guide tube that will provide a stable environment for axon proliferation while simultaneously preventing formation of fibrous scar tissue. Supplementing tubes with a collagen gel or collagen plus extracellular matrix factors is reported to increase axon proliferation when compared to controls. But there is no information regarding the use of collagen fibers to guide nerve cell migration through a tube. This communication reports ultrastructural observations on rat sciatic nerve regeneration through a silicone nerve stent containing crosslinked collagen fibers.Collagen fibers were prepared as described previously. The fibers were threaded through a silicone tube to form a central plug. One cm segments of sciatic nerve were excised from Sprague Dawley rats. A control group of rats received a silicone tube implant without collagen while an experimental group received the silicone tube containing a collagen fiber plug. At 4 and 6 weeks postoperatively, the implants were removed and fixed in 2.5% glutaraldehyde buffered by 0.1 M cacodylate containing 1.5 mM CaCl2 and balanced by 0.1 M sucrose. The explants were post-fixed in 1% OSO4, block stained in 1% uranyl acetate, dehydrated and embedded in Epon. Axons were counted on montages prepared at a total magnification of 1700x. Montages were viewed through a dissecting microscope. Thin sections were sampled from the proximal, middle and distal regions of regenerating sciatic plugs.

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