Tissue damage between barbed suture and conventional sutures in animal laboratory model using scanning electron microscopy.

This study aimed to compare the tissue damage caused by barbed sutures and conventional sutures using scanning electron microscopy (SEM). Porcine myocardium was incised and sutured using different thread types: barbed suture, (STRATAFIX® Spiral PDS PLUS) and conventional sutures, (VICRYL® and PDS Plus®). Needle hole shapes were examined at magnifications of 30×-100×. VICRYL® suture damaged the tissue and created large gaps around the needle holes. The tissue around the needle holes was smoother and less damaged in the single suture ligations with PDS®; however, a large gap had formed. In the continuous suture with STRATAFIX®, the tissue around the needle holes was significantly smoother and minimally damaged, with no noticeable gaps around the needle holes. Barbed sutures reduced the load on needle holes and minimised tissue damage owing to the dispersion of traction forces by the barbs compared with conventional sutures.

A new HPF specimen carrier adapter for the use of high-pressure freezing with cryoscanning electron microscope: two applications: stearic acid organization in a hydroxypropyl methylcellulose matrix and mice myocardium.

Cryogenic transmission electron microscopy of high-pressure freezing (HPF) samples is a well-established technique for the analysis of liquid containing specimens. This technique enables observation without removing water or other volatile components. The HPF technique is less used in scanning electron microscopy (SEM) due to the lack of a suitable HPF specimen carrier adapter. The traditional SEM cryotransfer system (PP3000T Quorum Laughton, East Sussex, UK; Alto Gatan, Pleasanton, CA, USA) usually uses nitrogen slush. Unfortunately, and unlike HPF, nitrogen slush produces water crystal artefacts. So, we propose a new HPF specimen carrier adapter for sample transfer from HPF system to cryogenic-scanning electronic microscope (Cryo-SEM). The new transfer system is validated using technical two applications, a stearic acid in hydroxypropyl methylcellulose solution and mice myocardium. Preservation of samples is suitable in both cases. Cryo-SEM examination of HPF samples enables a good correlation between acid stearic liquid concentration and acid stearic occupation surface (only for homogeneous solution). For biological samples as myocardium, cytoplasmic structures of cardiomyocyte are easily recognized with adequate preservation of organelle contacts and inner cell organization. We expect this new HPF specimen carrier adapter would enable more SEM-studies using HPF.

Cardiac Extracellular Matrix Modification as a Therapeutic Approach.

The cardiac extracellular matrix (cECM) is comprised of proteins and polysaccharides secreted by cardiac cell types, which provide structural and biochemical support to cardiovascular tissue. The roles of cECM proteins and the associated family of cell surface receptor, integrins, have been explored in vivo via the generation of knockout experimental animal models. However, the complexity of tissues makes it difficult to isolate the effects of individual cECM proteins on a particular cell process or disease state. The desire to further dissect the role of cECM has led to the development of a variety of in vitro model systems, which are now being used not only for basic studies but also for testing drug efficacy and toxicity and for generating therapeutic scaffolds. These systems began with 2D coatings of cECM derived from tissue and have developed to include recombinant ECM proteins, ECM fragments, and ECM mimics. Most recently 3D model systems have emerged, made possible by several developing technologies including, and most notably, 3D bioprinting. This chapter will attempt to track the evolution of our understanding of the relationship between cECM and cell behavior from in vivo model to in vitro control systems. We end the chapter with a summary of how basic studies such as these have informed the use of cECM as a direct therapy.

Ultrasonographic findings in goats with contagious caprine pleuropneumonia caused by Mycoplasma capricolum subsp. capripneumoniae.

BACKGROUND: In goats, contagious caprine pleuropneumonia (CCPP) is a cause of major economic losses in Africa, Asia and in the Middle East. There is no information emphasising the importance of diagnostic ultrasound in goats with CCPP caused by Mycoplasma capricolum subsp. capripneumoniae (Mccp). This study was designed to describe the ultrasonographic findings in goats with CCPP caused by Mccp and to correlate ultrasonographic with post-mortem findings. To this end, 55 goats with CCPP were examined. Twenty-five healthy adult goats were used as a control group. RESULTS: Major clinical findings included harried, painful respiration, dyspnoea and mouth breathing. On ultrasonography, a liver-like echotexture was imaged in 13 goats. Upon post-mortem examination, all 13 goats exhibited unilateral pulmonary consolidation. Seven goats had a unilateral hypoechoic pleural effusion. At necropsy, the related lung was consolidated and the pleural fluid appeared turbid and greenish. Pleural abscessiation detected in five goats was confirmed post-mortem. Twenty-eight goats had a bright, fibrinous matrix extending over the chest wall containing numerous anechoic fluid pockets with medial displacement and compression of lung tissue. Echogenic tags imaged floating in the fluid were found upon post-mortem examination to be fibrin. In two goats, a consolidated right parenchyma was imaged together with hypoechoic pericardial effusions with echogenic tags covering the epicardium. At necropsy, the right lung was consolidated in three goats and fibrin threads were found covering the epicardium and pericardium. CONCLUSIONS: In goats with CCPP, the extension and the severity of the pulmonary changes could not be verified with clinical certainty in most cases, whereas this was possible most of the time with sonography, thus making the prognosis easier. Ultrasonographic examination of the pleurae and the lungs helped in the detection of various lesions.

Nanoscale cues regulate the structure and function of macroscopic cardiac tissue constructs.

Heart tissue possesses complex structural organization on multiple scales, from macro- to nano-, but nanoscale control of cardiac function has not been extensively analyzed. Inspired by ultrastructural analysis of the native tissue, we constructed a scalable, nanotopographically controlled model of myocardium mimicking the in vivo ventricular organization. Guided by nanoscale mechanical cues provided by the underlying hydrogel, the tissue constructs displayed anisotropic action potential propagation and contractility characteristic of the native tissue. Surprisingly, cell geometry, action potential conduction velocity, and the expression of a cell-cell coupling protein were exquisitely sensitive to differences in the substratum nanoscale features of the surrounding extracellular matrix. We propose that controlling cell-material interactions on the nanoscale can stipulate structure and function on the tissue level and yield novel insights into in vivo tissue physiology, while providing materials for tissue repair.

The influence of repeated administration of poloxamer 407 on serum lipoproteins and protease activity in mouse liver and heart.

The effects of repeated administration of poloxamer 407 (P-407) on lipoprotein-cholesterol (LP-C) and lipoprotein-triglyceride (LP-TG) fractions and subfractions, as well as the effect on liver and heart proteases, were studied. Repeated administration of P-407 to male CBA mice resulted in a model of atherosclerosis with increased diastolic blood pressure; there was a drastic increase in total serum cholesterol and especially TG. A novel small-angle X-ray scattering method for the determination of the fractional and subfractional composition of LP-C and LP-TG was used. In chronically P-407-treated mice, P-407 significantly increased atherogenic low-density lipoprotein C (LDL-C) fractions, as well as intermediate-density lipoprotein C (IDL-C), and LDL1₋3-C subfractions, and very-low-density lipoprotein-C (VLDL-C) fractions, as well as VLDL1₋2-C and VLDL3₋5-C subfractions), to a lesser extent, the total anti-atherogenic high-density lipoprotein C (HDL-C) fraction, as well as HDL2-C and HDL3-C subfractions. Additionally, we demonstrated an increase in the serum chitotriosidase activity, without significant changes in serum matrix metalloprotease (MMP) activity. Morphological changes observed in P-407-treated mice included atherosclerosis in the heart and storage syndrome in the liver macrophages. P-407 significantly increased the activity of cysteine, aspartate proteases, and MMPs in the heart, and only the activity of cathepsin B and MMPs in the liver of mice. Thus, repeated administration of P-407 to mice induced atherosclerosis secondary to sustained dyslipidemia and formation of foamy macrophages in liver, and also modulated the activity of heart and liver proteases.

Development of the venous pole of the heart in the frog Xenopus laevis: a morphological study with special focus on the development of the venoatrial connections.

The heart of lung-breathing vertebrates normally shows an asymmetric arrangement of its venoatrial connections along the left-right (L-R) body axis. The systemic venous tributaries empty into the right atrium while the pulmonary venous tributaries empty into the left atrium. The ways by which this asymmetry evolves from the originally symmetrically arranged embryonic venous heart pole are poorly defined. Here we document the development of the venous heart pole in Xenopus laevis (stages 40-46). We show that, prior to the appearance of the mouth of the common pulmonary vein (MCPV), the systemic venous tributaries empty into a bilaterally symmetric chamber (sinus venosus) that is demarcated from the developing atriums by a circular ridge of tissue (sinu-atrial ridge). A solitary MCPV appears during stage 41. From the time point of its first appearance onwards, the MCPV lies cranial to the sinu-atrial ridge and to the left of the developing interatrial septum and body midline. L-R lineage analysis shows that the interatrial septum and MCPV both derive from the left body half. The CPV, therefore, opens from the beginning into the future left atrium. The definitive venoatrial connections are established by the formation of a septal complex that divides the lumen of the venous heart pole into systemic and pulmonary venous flow pathways. This complex arises from the anlage of the interatrial septum and the left half of the sinu-atrial ridge.

In situ mechanical analysis of myofibrillar perturbation and aging on soft, bilayered Drosophila myocardium.

Drosophila melanogaster is a genetically malleable organism with a short life span, making it a tractable system in which to study mechanical effects of genetic perturbation and aging on tissues, e.g., impaired heart function. However, Drosophila heart-tube studies can be hampered by its bilayered structure: a ventral muscle layer covers the contractile cardiomyocytes. Here we propose an atomic force microscopy-based analysis that uses a linearized-Hertz method to measure individual mechanical components of soft composite materials. The technique was verified using bilayered polydimethylsiloxane. We further demonstrated its biological utility via its ability to resolve stiffness changes due to RNA interference to reduce myofibrillar content or due to aging in Drosophila myocardial layers. This protocol provides a platform to assess the mechanics of soft biological composite systems and, to our knowledge, for the first time, permits direct measurement of how genetic perturbations, aging, and disease can impact cardiac function in situ.

Plug and seal: prevention of hypoxic cardiocyte death by sealing membrane lesions with antimyosin-liposomes.

The hallmark of cell death is the development of cell membrane lesions. Such lesions in the myocardium are usually associated with acute myocardial infarction. Minimizing myocardial necrosis by thrombolytic reperfusion therapy constitutes the only major treatment to date. We envisioned a method to seal these membrane lesions using immunoliposomes as a novel adjunctive approach. An antigen to intracellular cytoskeletal myosin in hypoxic embryonic cardiocytes is used as an anchoring site, and a specific antibody on immunoliposomes as the anchor to plug and to seal the membrane lesions. H9C2 cells were used because they are cardiocytes and are propagated in tissue culture and their viability may be assessed by various methods. Viability assessed by [3H]thymidine uptake in hypoxic cardiocyte cultures (n = 6 each) treated with antimyosin-immunoliposomes (3.26 +/- 0.483 x 10(6) c.p.m.) was similar to that of normoxic cells (3.68 +/- 0.328 x 10(6) c.p.m.), but was greater than those of untreated hypoxic cells (0.115 +/- 0.155 x 10(6) c.p.m.) or hypoxic cells treated with plain liposomes (1.140 +/- 0.577 x 10(6) c.p.m.). These results were reconfirmed by trypan blue exclusion and by fluorescent, confocal and transmission electron microscopy. They indicated that cell death in hypoxic cardiocytes can be prevented by targeted cell membrane sealing. This concept of cell salvage should be applicable in the prevention of cell death in different biological systems.

Characterization of perfusion decellularized whole animal body, isolated organs, and multi-organ systems for tissue engineering applications.

To expand the application of perfusion decellularization beyond isolated single organs, we used the native vasculature of adult and neonatal rats to systemically decellularize the organs of a whole animal in situ. Acellular scaffolds were generated from kidney, liver, lower limb, heart-lung system, and a whole animal body, demonstrating that perfusion decellularization technology is applicable to any perfusable tissue, independent of age. Biochemical and histological analyses demonstrated that organs and organ systems (heart-lung pair and lower limb) were successfully decellularized, retaining their extracellular matrix (ECM) structure and organ-specific composition, as evidenced by differences in organ-specific scaffold stiffness. Altogether, we demonstrated that organs, organ systems and whole animal bodies can be perfusion decellularized while retaining ECM components and biomechanics.

Accordion-like honeycombs for tissue engineering of cardiac anisotropy.

Tissue-engineered grafts may be useful in myocardial repair; however, previous scaffolds have been structurally incompatible with recapitulating cardiac anisotropy. Here, we use microfabrication techniques to create an accordion-like honeycomb microstructure in poly(glycerol sebacate), which yields porous, elastomeric three-dimensional (3D) scaffolds with controllable stiffness and anisotropy. Accordion-like honeycomb scaffolds with cultured neonatal rat heart cells demonstrated utility through: (1) closely matched mechanical properties compared to native adult rat right ventricular myocardium, with stiffnesses controlled by polymer curing time; (2) heart cell contractility inducible by electric field stimulation with directionally dependent electrical excitation thresholds (p<0.05); and (3) greater heart cell alignment (p<0.0001) than isotropic control scaffolds. Prototype bilaminar scaffolds with 3D interconnected pore networks yielded electrically excitable grafts with multi-layered neonatal rat heart cells. Accordion-like honeycombs can thus overcome principal structural-mechanical limitations of previous scaffolds, promoting the formation of grafts with aligned heart cells and mechanical properties more closely resembling native myocardium.

Myocardial remodelling induced by repeated low doses of isoproterenol.

In the present work, the effect of isoproterenol on the electrical properties of the rat heart and on the cytoarchitecture of the surviving cardiomyocytes was studied. Myocardial remodelling was induced by the daily administration of 5 mg/kg isoproterenol (Iso) for 7 days. Administration resulted in a significant increase (52%) in the ratio of left ventricular weight to body weight. ECG voltage criteria confirmed the presence of left ventricular hypertrophy. QT interval prolongation by 23% and 58% was found in Iso rats and in the corresponding isolated hearts, respectively. Spontaneously beating Iso hearts had a higher incidence of dysrhythmias. The surviving cardiomyocytes showed an irregular shape with cytoplasmic processes rich in ribosomes and rough endoplasmic reticulum. In these regions, myofibril disorganization and mitochondrial fission were observed. A greatly increased incidence of caveolae was seen in the plasma membrane and in the mouth of t-tubules. The membranes of t-tubules showed vesiculation, especially near the dyads. Repeated administration of isoproterenol led to hypertrophy, characterized by the existence of myocytes with simultaneous signs of both mature and postnatally developing cardiomyocytes. Structural microheterogeneities at the level of individual cells may represent one of the factors leading to electrical imbalance in the myocardial tissue remodelled by isoproterenol.

Extracellular Matrix from Whole Porcine Heart Decellularization for Cardiac Tissue Engineering.

Decellularization of whole porcine hearts followed by recellularization with fully differentiated cells made from patient-specific human induced pluripotent stem cells (iPSCs) may provide the ultimate solution for patients with heart failure. Decellularization is the process of completely disrupting all cells and removing the cellular components (e.g., antigenic proteins, lipids, DNA) from organic tissue, leaving only the extracellular matrix (ECM). The decellularization of porcine hearts can be accomplished in 24 h and results in 98% DNA removal with only 6 h of detergent exposure. Automatically controlling the pressure during decellularization reduces the detergent exposure time while still completely removing immunogenic cell debris.

Effect of collateral flow on epicardial and endocardial lysosomal hydrolases in acute myocardial ischemia.

Early changes in lysosomal enzymes must occur if their role is significant in irreversible myocardial injury. Therefore, we ligated the anterior descending coronary artery in 14 dogs and after 60 min excised epicardial and endocardial samples from the ischemic and adjacent normal heart. The collateral flow measured with radioactive microspheres in the endocardial samples averaged 19% of control. The muscle was disrupted and fractionated by ultracentrifugation into nuclear pellet (NP), heavy lysosomal pellet (HL), light lysosomal pellet (LL), microsomal pellet (M) and supernate (S). Electron microscopy demonstrated changes characteristic of sichemia in whole tissues and sedimented fractions. Acid phosphatase reaction product was present in residual bodies in the HL fraction and membrane-bound vesicles in the LL fraction and in the intact tissue. Significant decreases in the specific activity of N-acetyl-beta-glucosaminidase and beta-glucuronidase occurred in the endocardial LL fraction, while significant increases in both were found in the ts fraction (P less than 0.05). Losses of acid phosphatase occurred in both LL and S fractions. Moreover, decreases of total N-acetyl-beta-glucosaminidase in the HL fraction and of total beta-glucuronidase and acid phosphatase in the LL fraction were positively correlated (P less than 0.01) with the degree of ischemia measured with radioactive microspheres. Only insignificant enzymatic changes were found when the collateral flow was greater than 40%, and the differences were less significant in epicardial samples where the flow averaged 29%. The early loss of enzymes from the lysosomal fractions in severe ischemia suggests a role for lysosomal hydrolases in the necrosis that follows coronary occlusion.

Cardiac tissue Doppler imaging in sports medicine.

The differentiation of training-induced cardiac adaptations from pathological conditions is a key issue in sports cardiology. As morphological features do not allow for a clear delineation of early stages of relevant pathologies, the echocardiographic evaluation of left ventricular function is the technique of first choice in this regard. Tissue Doppler imaging (TDI) is a relatively recent method for the assessment of cardiac function that provides direct, local measurements of myocardial velocities throughout the cardiac cycle. Although it has shown a superior sensitivity in the detection of ventricular dysfunction in clinical and experimental studies, its application in sports medicine is still rare. Besides technical factors, this may be due to a lack in consensus on the characteristics of ventricular function in relevant conditions. For more than two decades there has been an ongoing debate on the existence of a supernormal left ventricular function in athlete's heart. While results from traditional echocardiography are conflicting, TDI studies established an improved diastolic function in endurance-trained athletes with athlete's heart compared with controls.The influence of anabolic steroids on cardiac function also has been investigated by standard echocardiographic techniques with inconsistent results. The only TDI study dealing with this topic demonstrated a significantly impaired diastolic function in bodybuilders with long-term abuse of anabolic steroids compared with strength-trained athletes without abuse of anabolic steroids and controls, respectively.Hypertrophic cardiomyopathy is the most frequent cause of sudden death in young athletes. However, in its early stages, it is difficult to distinguish from athlete's heart. By means of TDI, ventricular dysfunction in hypertrophic cardiomyopathy can be disclosed even before the development of left ventricular hypertrophy. Also, a differentiation of left ventricular hypertrophy due to hypertrophic cardiomyopathy or systemic hypertension is possible by TDI. Besides the evaluation of different forms of left ventricular hypertrophy, the diagnosis of myocarditis is also of particular importance in athletes. Today, it still requires myocardial biopsy. The analysis of focal disturbances in myocardial velocities might be a promising non-invasive method; however, systematic validation studies are lacking. An important future issue for the implementation of TDI into routine examination will be the standardisation of procedures and the establishment of significant reference values for the above-mentioned conditions. Innovative TDI parameters also merit further investigation.

Pharmacological, toxicological, and therapeutic evaluation in mice of doxorubicin entrapped in cardiolipin liposomes.

Doxorubicin possesses high affinity for binding to cardiolipin. We have utilized these properties in preparing stable liposomes of doxorubicin and cardiolipin with a net positive charge. Doxorubicin liposomes were formed by using 11.2 mumol of drug, 5.6 mumol of cardiolipin, 28.5 mumol of phosphatidylcholine, 19.5 mumol of cholesterol, and 11.1 mumol of stearylamine. These liposomes were sonicated for 90 min at 37 degrees followed by extensive dialysis against buffer. The pharmacological, toxicological, and therapeutic effects of doxorubicin entrapped in cardiolipin liposomes were compared with those of free doxorubicin in mice. At a dose of 4 mg/kg i.v., the peak cardiac concentration was achieved in 30 min following free doxorubicin administration, the value being 8.1 micrograms/g. The peak cardiac concentration with doxorubicin in cardiolipin liposomes was obtained at 5 min with a value of 2.8 micrograms/g of tissue. The cardiac concentration X time values for free doxorubicin for the 24-hr period of observation were 55.1 micrograms X hr/g, whereas it was only 7.8 micrograms X hr/g with the drug entrapped in cardiolipin liposomes. Compared to free drug, the liposomal entrapped doxorubicin significantly reduced the histopathological lesions in cardiac tissue of mice at a dose of 15 mg/kg as determined by electron microscopy. The nadir of peripheral white blood cell counts in mice with free drug, 6 mg/kg, was observed on Day 3 which was 50% of control, whereas with liposomal encapsulated drug it was reduced only 23% on Day 7. Doxorubicin in cardiolipin liposomes demonstrated enhanced chemotherapeutic potential against murine ascitic P388 leukemia with a 144% increased life span compared to 55% increased life span with free drug at a dose of 7.5 mg/kg on Days 1, 3, and 7. We conclude that doxorubicin liposomes developed in these studies possess improved therapeutic action as demonstrated by their ability to reduce the toxicity of the drug substantially.

The cutting of ultrathin sections with the thickness less than 20 nm from biological specimens embedded in resin blocks.

Low voltage electron microscopes working in transmission mode, like LVEM5 (Delong Instruments, Czech Republic) working at accelerating voltage 5 kV or scanning electron microscope working in transmission mode with accelerating voltage below 1 kV, require ultrathin sections with the thickness below 20 nm. Decreasing of the primary electron energy leads to enhancement of image contrast, which is especially useful in the case of biological samples composed of elements with low atomic numbers. As a result treatments with heavy metals, like post-fixation with osmium tetroxide or ultrathin section staining, can by omitted. The disadvantage is reduced penetration ability of incident electrons influencing the usable thickness of the specimen resulting in the need of ultrathin sections of under 20 nm thickness. In this study we want to answer basic questions concerning the cutting of extremely ultrathin sections: Is it possible routinely and reproducibly to cut extremely thin sections of biological specimens embedded in commonly used resins with contemporary ultramicrotome techniques and under what conditions? Microsc. Res. Tech. 79:512-517, 2016. © 2016 Wiley Periodicals, Inc.

Single oral acute fluoride exposure causes changes in cardiac expression of oxidant and antioxidant enzymes, apoptotic and necrotic markers in male rats.

Several studies have shown that acute fluoride (F(-)) exposure impairs cardiac function, but the molecular mechanism is not clear. In order to study this, male Wistar rats were treated with single oral doses of 45 and 90 mg/kg F(-) for 24 h. A significant accumulation of F(-) was found in the serum and myocardium of experimental rats. F(-) treatment causes myocardial necrosis as evident from increased levels of myocardial troponin I, creatine kinase, lactate dehydrogenase and aspartate transaminase. In addition, F(-) induces myocardial oxidative stress via increased reactive oxygen species, lipid peroxidation, protein carbonyl content and nitrate levels along with decreased in the levels of enzymatic (superoxide dismutase 2, catalase, glutathione peroxidase and glutathione s transferase pi class) and non-enzymatic (reduced glutathione) antioxidants. Notably, F(-) triggers myocardial apoptosis through altered Bax/Bcl2 ratio and increased cytochrome c, caspase 3p20 and terminal deoxynucleotidyl transferase dUTP nick end labeled positive cells. An increased cardiac expression of Nox4 and p38α MAPK in F(-) treated rats indicates the oxidative and apoptotic damage. Moreover, ultra-structural changes, histopathological and luxol fast blue staining demonstrates the degree of myocardial damage at subcellular level. Taken together, these findings reveal that acute F(-) exposure causes cardiac impairment by altering the expression of oxidative stress, apoptosis and necrotic markers.

Ultrastructural distribution of myosin heavy chain mRNA in cardiac tissue: a comparison of frozen and LR White embedment.

Electron microscopy (EM) in situ hybridization provides the higher resolution necessary to determine the spatial relationship between a specific mRNA and the organelle containing the protein encoded by that message. EM in situ hybridization was used to determine the subcellular myosin heavy chain (MHC) mRNA distribution with respect to the myofibril in normal cardiac tissue. Sections of frozen or acrylic-embedded tissue were compared for ultrastructural integrity and content of endogenous mRNA. Papillary muscles dissected from hearts of normal rabbits were aldehyde-fixed and either frozen or embedded in LR White. EM in situ hybridization with no riboprobe, vector sequence, same-sense, and anti-sense biotinylated riboprobes was detected by indirect immunocytochemistry. Labeling density using an antisense probe was highest over the intermyofibrillar space, with an average signal five times that of background. Background labeling by nonspecific sense probe was consistently low but not random, also having the highest density of gold clusters over the intermyofibrillar space. Ultracryomicrotomy yielded a higher absolute number of gold clusters, but sections were fragmented and disrupted striated muscle morphology. LR White embedment maintained ultrastructural integrity but gave a lower absolute signal. Fortunately, MHC mRNA is an abundant message and can tolerate the decreased sensitivity of LR White.

Immunoelectron microscopic localization of alpha-actinin on Lowicryl-embedded thin-sectioned tissues.

A procedure has been developed for the immunoelectron microscopic localization of intracellular antigens on thin-sectioned tissues. The tissues were fixed in a periodate-lysine-paraformaldehyde solution or a formaldehyde-glutaraldehyde combination and embedded in the acrylate-methacrylate mixture, Lowicryl K4M (Polaron), which was polymerized under ultraviolet irradiation at -35 degrees C. Thin sections were mounted on gold grids, immunostained using an indirect method with ferritin-labeled antibodies, and, optionally, counterstained with osmium tetroxide and/or lead citrate and uranyl acetate. The procedure provided good morphologic preservation of the cell architecture in adult and embryonic heart, and skeletal and smooth muscle tissue, as well as nonmuscle cells. At the same time it retained the antigenicities of several contractile proteins, including myosin, tropomyosin, actin, and alpha-actinin. The method has advantages over en bloc staining techniques in that the problem of antibody penetration into the cells is eliminated and careful controls can be performed on adjacent sections. This technique will be useful for localizing, at the ultrastructural level, contractile and other selected proteins in a variety of muscle and non-muscle cells. Details of the new protocol and a description of the results of using antibody against the contractile protein, alpha-actinin, are given.