[16] Immunohistochemical techniques to study the extracellular matrix and its receptors

Complete antigen visualization in the context of well-preserved tissue architecture is the goal of all immunohistochemical techniques. Frozen tissue section techniques achieve optimal antigen visualization but preserve tissue architecture poorly. On the other hand, formalin-fixed tissue section techniques preserve tissue architecture very well but result in antigen masking. Enzymatic digestion or salt extraction of formalin-fixed sections has been used to reestablish antigen expression. Recently acid-alcohol-fixed tissue has been used as a successful compromise between tissue architecture preservation and the visualization of cytoskeletal antigens. In an attempt to find an improved immunohistochemical process for non-cytoskeletal antigens, we compared avidin-biotin immunofluorescence staining in frozen, formalin-fixed, and acid-alcohol-fixed tissues. The fixed tissues were either untreated or treated with enzyme digestion or salt extraction. For this study, we examined healing cutaneous wounds in Yorkshire pigs with antibodies to fibronectin, laminin, von Willebrand factor VIII, and keratin. Although tissue architecture was poor, frozen sections provided the best antigen visualization and were therefore used as the standard for complete antigen expression. Formalin-fixed tissues had excellent tissue architecture, but most antigens were completely masked. Pre-treatment technique only partially overcame the antigen masking caused by formalin. In contrast, acid-alcohol fixation preserved tissue architecture almost as well as formalin and sometimes allowed complete antigen visualization; however, laminin and fibronectin were partially masked. Total recovery of the expression of these antigens could be obtained by pre-treating the acid-alcohol-fixed tissue with either hyaluronidase or 1 M NaCl. Therefore, acid-alcohol-fixed tissue appears best for extracellular matrix (ECM) protein immunostaining as well as for cytoskeletal staining. However, certain ECM antigens require hyaluronidase or 1 M NaCl treatment for optimal visualization.

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Immunohistochemical Analysis of Type III and IV Collagens in Tubulointerstitial Damage in Human Benign Nephrosclerosis

Journal of International Medical Research ◽

10.1177/030006059502300610 ◽

1995 ◽

Vol 23 (6) ◽

pp. 480-486 ◽

Cited By ~ 4

Author(s):

M S Razzaque ◽

M Cheng ◽

Y Horita ◽

M Nishihara ◽

T Harada ◽

...

Keyword(s):

Extracellular Matrix ◽

Basement Membrane ◽

Structural Changes ◽

Immunohistochemical Analysis ◽

Type Iii Collagen ◽

Type Iii ◽

Type Iv ◽

Tubulointerstitial Damage ◽

Basement Membrane Collagen ◽

Immunohistochemical Techniques

Prolonged hypertension causes structural changes including glomerulosclerosis and tubulointerstitial damage of the kidney, termed benign nephrosclerosis. It is generally accepted that, in benign nephrosclerosis, increased accumulation of extracellular matrix in the glomeruli results in glomerulosclerosis. Little is known, however, about the possible role of the extracellular matrix in the tubulointerstitial damage in benign nephrosclerosis. In this study, the possible roles of type IV basement-membrane collagen and type III interstitial collagen in tubulointerstitial damage caused by hypertension were explored. Immunohistochemical techniques were used to investigate the distribution of type III and type IV collagens in the kidney sections of 15 patients with benign nephrosclerosis with tubulointerstitial damage and in 10 controls. In the control renal sections strong immunostaining for type III collagen was found in the interstitium and immunostaining for type IV collagen was present in the tubular basement membrane and weakly in the interstitium. In the patients with tubulointerstitial damage there was increased immunostaining for both type III and type IV collagens in the expanded interstitium and damaged tubules than was found in the control kidney sections. These findings indicate that increased accumulation of both type III and type IV collagens might play a significant role in the tubulointerstitial damage in benign nephrosclerosis.

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Immunohistochemical Detection of Apolipoprotein A-I and B-100 in Canine Atherosclerotic Lesions

Veterinary Pathology ◽

10.1354/vp.40-3-328 ◽

2003 ◽

Vol 40 (3) ◽

pp. 328-331 ◽

Cited By ~ 4

Author(s):

T. Sako ◽

E. Uchida ◽

Y. Kagawa ◽

K. Hirayama ◽

T. Nakade ◽

...

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Coronary Arteries ◽

Peripheral Arteries ◽

Immunohistochemical Detection ◽

Atherosclerotic Lesions ◽

Apolipoprotein A ◽

Subendothelial Space ◽

Immunohistochemical Techniques ◽

Tunica Intima

We attempt to determine and compare the localization of apolipoproteins (apo) apoA-I and B-100 in atherosclerotic lesions of canine aortas, coronary arteries, and the peripheral arteries, using immunohistochemical techniques. Histopathologically, atherosclerotic lesions were characterized by deposition of lipids and infiltration of lipid-laden foamy cells in the tunica intima and tunica media, sometimes forming fibrofatty plaques containing abundant sudanophilic and mineralized material. Canine apoA (CapoA)-I and canine apoB (CapoB)-100 immunopositive signals were simultaneously observed in mild and severe atherosclerotic lesions of the aorta, coronary arteries, splenic arteries, and renal arteries in the double-immunolabeled sections. Both CapoAI and CapoB-100 positive signals were seen in the cytoplasm of endothelial cells, smooth muscle cells, and macrophages. The subendothelial space and extracellular matrix in the tunica intima and media were also positive. Neither CapoA-I nor CapoB-100 positive signals were seen in normal arteries. These findings closely resemble those of the localization of apoA-I and apoB-100 in human atherosclerotic lesions.

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Tenascin is synthesized and secreted by rat mesangial cells in culture and is present in extracellular matrix in human glomerular diseases.

Journal of the American Society of Nephrology ◽

10.1681/asn.v4101771 ◽

1994 ◽

Vol 4 (10) ◽

pp. 1771-1777

Author(s):

L D Truong ◽

M W Majesky ◽

J Pindur

Keyword(s):

Extracellular Matrix ◽

Mesangial Cells ◽

Kidney Tissue ◽

Distinct Species ◽

Renal Diseases ◽

Northern Hybridization ◽

Collagen Type Iv ◽

Mesangial Matrix ◽

Glomerular Diseases ◽

Immunohistochemical Techniques

Tenascin (TN) is a large oligomeric protein recently described as a component of the extracellular matrix. The distribution of TN in adult kidney tissue has not been adequately evaluated, but preliminary data have suggested that TN is variably seen in rare mesangial areas and in stroma surrounding some tubules. The enlargement of the mesangial matrix (mesangial sclerosis) is a common feature of many renal diseases and is thought to be partially related to oversynthesis of the normal components of the mesangial matrix (collagen type IV, laminin, fibronectin, and heparan sulfate proteoglycans) by mesangial cells. However, the possibility that mesangial cells are also the source of other extracellular matrix proteins that participate in the process of mesangial sclerosis has not been explored. In this study, the synthesis of TN by cultured rat mesangial cells was documented by the following observations: (1) Northern hybridization of total RNA extracted from mesangial cells showed two distinct species of TN mRNA; (2) immunoblotting of the protein extracted from the conditioned medium demonstrated four TN protein bands; (3) immunoblotting of the protein extracted from the mesangial cell lysate demonstrated at least four TN protein bands; and (4) immunohistochemical techniques identified TN within the cytoplasm of mesangial cells and in the surrounding extracellular matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

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Effect of spaceflight on the extracellular matrix of skeletal muscle after a crush injury

Journal of Applied Physiology ◽

10.1152/jappl.1992.73.2.s74 ◽

1992 ◽

Vol 73 (2) ◽

pp. S74-S81 ◽

Cited By ~ 11

Author(s):

W. T. Stauber ◽

V. K. Fritz ◽

T. E. Burkovskaya ◽

E. I. Ilyina-Kakueva

Keyword(s):

Skeletal Muscle ◽

Extracellular Matrix ◽

Chronic Inflammation ◽

Muscle Regeneration ◽

Gastrocnemius Muscle ◽

Matrix Material ◽

Repair Process ◽

Treatment Groups ◽

Nonmuscle Cells ◽

Immunohistochemical Techniques

The organization and composition of the extracellular matrix were studied in the crush-injured gastrocnemius muscle of rats subjected to 0 G. After 14 days of flight on COSMOS 2044, the gastrocnemius muscle was removed and evaluated by histochemical and immunohistochemical techniques from the five injured flight rodents and various Earth-based treatment groups. In general, the repair process was similar in all injured muscle samples with regard to the organization of the extracellular matrix and myofibers. Small and large myofibers were present within an expanded extracellular matrix, indicative of myogenesis and muscle regeneration. In the tail-suspended animals, a more complete repair was observed with no enlarged area of nonmuscle cells or matrix material visible. In contrast, the muscle samples from the flight animals were less well organized and contained more macrophages and blood vessels in the repair region, indicative of a delayed repair process, but did not demonstrate any chronic inflammation. Myofiber repair did vary in muscles from the different groups, being slowest in the flight animals and most complete in the tail-suspended ones.

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Cardiac myocytes cultured on a basement membrane extract of the ehs tumor

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142979 ◽

1986 ◽

Vol 44 ◽

pp. 270-271

Author(s):

L. Terracio ◽

A. Dewey ◽

K. Rubin ◽

T.K. Borg

Keyword(s):

Extracellular Matrix ◽

Basement Membrane ◽

Cardiac Myocytes ◽

Normal Tissue ◽

Cell Spreading ◽

Collagen Iv ◽

Basement Membrane Extract ◽

Membrane Extract ◽

Growth Development ◽

Multicellular Organisms

The recognition and interaction of cells with the extracellular matrix (ECM) effects the normal physiology as well as the pathology of all multicellular organisms. These interactions have been shown to influence the growth, development, and maintenance of normal tissue function. In previous studies, we have shown that neonatal cardiac myocytes specifically interacts with a variety of ECM components including fibronectin, laminin, and collagens I, III and IV. Culturing neonatal myocytes on laminin and collagen IV induces an increased rate of both cell spreading and sarcomerogenesis.

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Neutrophil migration through in vitro interstitial matrix

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100124872 ◽

1992 ◽

Vol 50 (1) ◽

pp. 894-895

Author(s):

J. Roemer ◽

S.R. Simon

Keyword(s):

Extracellular Matrix ◽

Smooth Muscle ◽

Cell Migration ◽

Smooth Muscle Cells ◽

Inflammatory Cell ◽

Neutrophil Migration ◽

Culture Conditions ◽

Aortic Smooth Muscle ◽

Specific Culture

We are developing an in vitro interstitial extracellular matrix (ECM) system for study of inflammatory cell migration. Falcon brand Cyclopore membrane inserts of various pore sizes are used as a support substrate for production of ECM by R22 rat aortic smooth muscle cells. Under specific culture conditions these cells produce a highly insoluble matrix consisting of typical interstitial ECM components, i.e.: types I and III collagen, elastin, proteoglycans and fibronectin.

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The extracellular matrix of echinoderm and amphibian eggs: Visualization in quick-frozen, deep-etched, and rotary-shadowed specimens

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010015784x ◽

1990 ◽

Vol 48 (3) ◽

pp. 60-61

Author(s):

Barry Bonnell ◽

Carolyn Larabell ◽

Douglas Chandler

Keyword(s):

Extracellular Matrix ◽

Plasma Membrane ◽

Sea Urchin ◽

Crystalline Structure ◽

Cortical Granule ◽

Two Dimensional ◽

Small Peptides ◽

Deep Etching ◽

Quick Freezing ◽

Acrosomal Reaction

Eggs of many species including those of echinoderms, amphibians and mammals exhibit an extensive extracellular matrix (ECM) that is important both in the reception of sperm and in providing a block to polyspermy after fertilization.In sea urchin eggs there are two distinctive coats, the vitelline layer which contains glycoprotein sperm receptors and the jelly layer that contains fucose sulfate glycoconjugates which trigger the acrosomal reaction and small peptides which act as chemoattractants for sperm. The vitelline layer (VL), as visualized by quick-freezing, deep-etching, and rotary-shadowing (QFDE-RS), is a fishnet-like structure, anchored to the plasma membrane by short posts. Orbiting above the VL are horizontal filaments which are thought to anchor the thicker jelly layer to the egg. Upon fertilization, the VL elevates and is transformed by cortical granule secretions into the fertilization envelope (FE). The rounded casts of microvilli in the VL are transformed into angular peaks and the envelope becomes coated inside and out with sheets of paracrystalline protein having a quasi-two dimensional crystalline structure.

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Extracellular matrix: the gatekeeper of tumor angiogenesis

Biochemical Society Transactions ◽

10.1042/bst20190653 ◽

2019 ◽

Vol 47 (5) ◽

pp. 1543-1555 ◽

Cited By ~ 10

Author(s):

Maurizio Mongiat ◽

Simone Buraschi ◽

Eva Andreuzzi ◽

Thomas Neill ◽

Renato V. Iozzo

Keyword(s):

Extracellular Matrix ◽

Tumor Angiogenesis ◽

Signaling Cascades ◽

Cancer Growth ◽

Cell Behavior ◽

Metastatic Progression ◽

Surface Receptors ◽

The Matrix ◽

Multiple Signaling

Abstract The extracellular matrix is a network of secreted macromolecules that provides a harmonious meshwork for the growth and homeostatic development of organisms. It conveys multiple signaling cascades affecting specific surface receptors that impact cell behavior. During cancer growth, this bioactive meshwork is remodeled and enriched in newly formed blood vessels, which provide nutrients and oxygen to the growing tumor cells. Remodeling of the tumor microenvironment leads to the formation of bioactive fragments that may have a distinct function from their parent molecules, and the balance among these factors directly influence cell viability and metastatic progression. Indeed, the matrix acts as a gatekeeper by regulating the access of cancer cells to nutrients. Here, we will critically evaluate the role of selected matrix constituents in regulating tumor angiogenesis and provide up-to-date information concerning their primary mechanisms of action.

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