Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.     

Localization of Proanthocyanidins Using in Situ-Hydrolysis with Sulfuric Acid

A valuable method for specific localization of proanthocyanidins in plant tissues is given. The specificity is based on the acid hydrolysis of condensed tannins in situ via hot sulfuric acid, yielding bright red anthocyanidins. This treatment did not cause noticeable damage of tissue embedded in glycol methacrylate prior to sectioning. Further proof of specificity was achieved by control staining with the flavan-3-01-selective p-dimethylaminocinnamaldehyde (DMACA) reagent. In addition, comparison of adjacent sections stained with the DMACA reagent may give coarse information about the degree of polymerization of the proanthocyanidins.     

Nitrofurazone-induced DNA damage to tissues of mice

Cytotoxicity and DNA damage by nitrofurans has previously been correlated with metabolic reduction of these drugs in vitro. In the present study, nitrofurazone increased the rate of disappearance of stable [³H]thymidine labelled DNA from tissues of mice fed 0.1% nitrofurazone in the diet. Significant loss of labelled DNA occurred within 25 days after the start of the diet in all tissue observed, and loss was in relation to the rate of metabolic reduction of nitrofurazone. A similar correlation was found when another endpoint for DNA damage was used; nitrofurazone reduced by mouse tissue slices caused DNA single-strand breaks in cultured mouse L cells incubated in vitro with the tissues. Again, the ability of each tissue to produce toxic nitrofurazone metabolites determined the amount of DNA damage to the L cells.     

A new physiological phenomenon of mammalian body for organ and tissue neo-regeneration in vivo: adult stem cell technology in the perspective of literature

A new phenomenon by which adult developed mammalian and human body, can neo-regenerate its own tissue/organ in vivo, is recognized as "Desired Metaplasia". Reserve stem cells present in the tissues of adult developed body, are responsible for repair and replacement of lost tissues and cells during life, is plasia. Chronic tissue damage, due to long-standing causative factor, is taken care of and protected by forming resistant tissues, is metaplasia. Both these changes take place at the anatomical abode of the tissues. When stem cells of one tissue are colonized with another tissue/organ system, away from its anatomical abode, the neo-organo-histogenesis takes place by a phenomenon of desired metaplasia. This being new is critically, analytically and scientifically studied and discussed with reference to the available literature. An attempt has been made to establish the scientific account of the phenomenon. The laws of nature and embryology, as well as the basic philosophy responsible for neo-regeneration of tissues and organs in vivo, are discussed. In conclusion, the mammalian and human bodies can neo-regenerate their tissues and organs in vivo by desired metaplasia, provided certain criteria of embryological organogenesis are strictly observed.     

In vivo gene gun-mediated DNA delivery into rodent brain tissue

Various types of gene transfer into live tissues have been tried. However, in vivo gene transfer into brain tissue or neuronal cells without virus vector has required a great effort. Particle-mediated gene transfer into live brain tissue was thought to be impossible because of its fragility and the mechanical problem of a previous type of gene gun. In addition, particle-mediated DNA transfer into monolayer-cultured cells without mechanical damage has been difficult. We successfully transferred DNA into rodent live brain tissue and also into monolayer-cultured cells without mechanical damage by using a new type of gene gun and also confirmed gene expression in the brain. This new method represents another variation of gene transfer into the brain.     

Issues in stem cell plasticity

Experimental biology and medicine work with stem cells more than twenty years. The method discovered for in vitro culture of human embryonal stem cells acquired at abortions or from?surplus” embryos left from in vitro fertilization, evoked immediately ideas on the posibility to aim development and differentiation of these cells at regeneration of damaged tissues. Recently, several surprising observations proved that even tissue‐specific (multipotent) stem cells are capable, under suitable conditions of producing a while spectrum of cell types, regardless, whether these tissues are derived from the same germ layer or not. This ability is frequently called stem cell plasticity but other authors also use different names ‐?non‐orthodox differentiation” or?transdifferentiation”. In this paper we wish to raise several important questions and problems related to this theme. Let us remind some of them: Is it possible to force cells of one‐type tissue to lool and act as cells of another tissue? Are these changes netural? Could these trans‐formations be used to treat diseases? What about the bioethic issue? However, the most serious task “still remains to be soloved ‐ how to detect, harvestand culture stem cells for therapy of certain diseases”.     

Mammalian pyruvate kinase hybrid isozymes: tissue distribution and physiological significance

The purpose of this study was to examine the pyruvate kinase isozymic patterns of a wide variety of tissues from rats and mice, particularly regarding hybrid isozymes. For these studies, we employed longer electrophoresis times than used in most earlier studies in order to improve the resolution of closely spaced bands. The tissue distributions of types K, L, and M pyruvate kinases were found to be approximately the same as those reported earlier for rats and other mammals. In addition, K-M hybrids could be detected in most tissues examined in relative quantities which differed from one tissue to another in the same organism, in corresponding tissues from different species, and within a single tissue during development. Hybrid isozymes containing type L subunits occur in only a few tissues of either the fetus or the adult of either animal. In earlier studies utilizing L-M hybrid isozymes produced in vitro, we showed that the kinetic properties of a given subunit are profoundly affected by the nature of its neighbors within the tetramer (Dyson and Cardenas, ['73] J. Biol. Chem., 248: 8482-8488). Based on these altered kinetic properties, we suggest that there is little need for anorganism to suppress completely the gene activity for one subunit type of pyruvate kinase during the synthesis of larger quantities of a second subunit type.     

Freezing for postmortal storage influences the biomechanical properties of linear skin wounds.

Specimens for biomechanical investigations are often stored frozen between sampling and testing. Several authors have analysed the effects of freezing on a variety of intact tissues; while some have found mostly minor changes, others have reported no adverse effects. Healing wounds contain more fragile tissue components than other tissues and are therefore more sensitive to possible adverse effects. This study on rat skin wounds (healed for 10 and 20 days) demonstrates that freezing has a significant adverse influence on the mechanical properties. It is concluded that fresh tissue should be used whenever possible. In case storage in a freezer is necessary great care should be taken when designing the experimental protocol.     

Octreotide ameliorates sepsis-induced pelvic inflammation in female rats by a neutrophil-dependent mechanism

Sepsis is a generalized inflammatory response, which involves organ systems remote from the locus of the initial infectious insult, accompanied by the release of cytokines and the subsequent formation of reactive oxygen and nitrogen species. The aim of this study was to investigate the possible protective effect of octreotide (OCT), a synthetic somatostatin analogue, against sepsis-induced oxidative damage in the uterine and ovarian tissues of rats. Sepsis was induced by caecal ligation and puncture method in female Wistar albino rats. Sepsis and sham operated (control) groups received either saline or OCT (50 microg/kg, i.p.; Novartis) immediately after the operation and at 12 h. Twenty-four hours after the surgery, rats were decapitated and serum TNF-alpha levels and tissue malondialdehyde (MDA) content, glutathione (GSH) levels and myeloperoxidase (MPO) activity were determined in the uterus and ovaries. Oxidant-induced tissue fibrosis was determined by tissue collagen contents, while the extent of tissue injuries was analyzed microscopically. Sepsis increased serum TNF-alpha levels and resulted in decreased GSH levels and increased MDA levels, MPO activity and collagen contents in both the uterus and the ovaries (p<0.05-0.001) indicating the presence of the oxidative damage, as also confirmed by histological analysis. On the other hand, OCT administration reversed these oxidant responses and reduced the severity of microscopic damage (p<0.001). In conclusion, OCT protects against sepsis-induced oxidative injury of the uterine and ovarian tissues by diminishing neutrophil infiltration, an important source of oxygen free radicals. Our results suggest that OCT may be of therapeutic value in ameliorating sepsis-associated pelvic inflammation.     

应用冷Schiff组织化学法检测人体组织脂质过氧化反应的研究

自由基引发的生物膜不饱和脂肪酸脂质过氧化反应涉及多种疾病过程,多年来检测脂质过氧化反应一直沿用生物化学（如硫代巴比妥酸法测定丙二醛）或生物物理技术（如分光光度法测定共轭双烯）。自从冷Schiff组织化学染色技术用于自由基研究以来,使形态学方法研究脂质过氧化反应成为可能,当前,应用冷Schiff组织化学法进行组织细胞的脂质过氧化反应检测大多限于动物实验研究,本研究对多种人体离体新鲜组织的冰冻切片应用冷Schiff组织化学法进行检测,未发现被组织存在组织化学水平上的脂质过氧化反应;胆对被测组织人为施加氧化攻击（用Fe-NADPH促氧化剂孵育）后,肝、肾及胃的泌酸细胞与其它组织相比呈现较明显的脂质过氧化反应;皮肤、脂肪组织几科不出现脂质过氧化反应;甲状腺C细胞、肌肉、骨骼等与钙代谢、贮存及利用相关的组织也出现较明显的脂质过氧化反应。结论：冷Schiff组织化学方法检测人体组织脂持过氧化反应具有简便易行、同时可以形态学定位的优点,在医学生物学、肿瘤学及老年医学研究中具有应用前景。     

Effects of pertussis and cholera toxin on the interferon-γ stimulated immunocytochemical staining of ICAM-1 and inositol phosphate formation in a human renal carcinoma cell line

This report describes a new method using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to visualize live viable tissue at the microscopic level. The visualization of the MTT stained tissue provides a metabolic assessment of tissue damage, and can be utilized as an extension of conventional hematoxylin-eosin (H & E) staining. In this report, several tissues were studied with MTT and H & E staining after incisions had been made by a variety of surgical dissecting instruments. A marked improvement in the detection of tissue damage was seen using MTT, regardless of how the damage was caused, i.e., physical, heat, or photon energy. In addition, a distinct zone of damage not noted on conventionally prepared and stained tissues is readily apparent. Thus MTT staining will have utility in both clinical and research studies, concerned with assessing the viability of tissues.     

基因组织特异性相关研究进展

研究基因的组织特异性是了解生命活动进程和组织功能的重要一步.尽管对于看家基因和组织特异基因的研究由来已久,但是对于它们仍缺少统一的定义方式和检测方法.在定义方式上,可以从基因的组织表达数和在各组织间的表达变化情况来分别定义看家基因和组织特异基因.通常将在大多数正常组织中有表达,且表达水平较稳定的基因称为看家基因,而将在一个或少数组织中优势表达的基因定义为组织特异基因或组织选择基因.在检测方法上,高通量实验技术,包括基因芯片、RNA-seq和质谱技术等已成为检测基因组织特异性的主要方法.通过比较多个典型研究的实验结果,发现不同检测方法的覆盖度和灵敏度存在很大差异,其中RNA-seq技术最为灵敏,获得的看家基因数目最多,质谱技术检测出来的看家基因和组织特异基因数目较少,而基因芯片方法给出的多个检测结果间差别较大.尽管不同的定义方式和检测方法所导致的看家基因(或组织特异基因)的集合不完全一致,但不同的看家基因数据集(或组织特异基因)却展现出非常一致的功能和特性.看家基因通常实现所有组织和细胞都必须的基本功能,而看家基因与其他组织表达基因间的相互作用以及组织特异基因间的相互作用则实现了组织的特有功能.同时,基因的组织特异性与疾病之间具有密切联系,相比其他基因,看家基因更有可能成为癌基因,而组织特异基因则更有希望发展成为药物靶标.     

Comparison of two PCR methods for detection of Leptospira interrogans in formalin-fixed and paraffin-embedded tissues

In this study we compared two polymerase chain reaction (PCR) methods using either 16S ribosomal RNA (rRNA) or 23S rRNA gene primers for the detection of different Leptospira interrogans serovars. The performance of these two methods was assessed using DNA extracted from bovine tissues previously inoculated with several bacterial suspensions. PCR was performed on the same tissues before and after the formalin-fixed, paraffin-embedding procedure (FFPE tissues). The 23S rDNA PCR detected all fresh and FFPE positive tissues while the 16S rDNA-based protocol detected primarily the positive fresh tissues. Both methods are specific for pathogenic L. interrogans. The 23S-based PCR method successfully detected Leptospira in four dubious cases of human leptospirosis from archival tissue specimens and one leptospirosis-positive canine specimen. A sensitive method for leptospirosis identification in FFPE tissues would be a useful tool to screen histological specimen archives and gain a better assessment of human leptospirosis prevalence, especially in tropical countries, where large outbreaks can occur following the rainy season.     

Detection of abasic sites and oxidative DNA base damage using an ELISA-like assay

Reactive oxygen species produce a wide spectrum of DNA damage, including oxidative base damage and abasic (AP) sites. Many procedures are available for the quantification and detection of base damage and AP sites. However, either these procedures are laborious or the starting materials are difficult to obtain. A biotinylated aldehyde-specific reagent, ARP, has been shown to react specifically with the aldehyde group present in AP sites, resulting in biotin-tagged AP sites in DNA. The biotin-tagged AP sites can then be determined colorimetrically with an ELISA-like assay, using avidin/biotin-conjugated horseradish peroxidase as the indicator enzyme. The ARP assay is thus a simple, rapid, and sensitive method for the detection of AP sites in DNA. Furthermore, removal of damaged base by DNA N-glycosylases generates AP sites that can be measured by the ARP reagent. By coupling the ARP assay with either endonuclease III from Escherichia coli or 8-oxoguanine N-glycosylase (OGG1) from yeast, investigators can rapidly determine the amount of oxidative pyrimidine damage (endonuclease III-sensitive sites) or purine damage (OGG1-sensitive sites) in cellular DNA, respectively. An increased level of oxidative damage has been implicated in several age-related human diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease, as well as the aging process. The sensitivity and simplicity of the ARP assay thus make it a valuable method for investigators who are interested in estimating the level of oxidative DNA damage in cells and tissues derived from patients with various age-related diseases or cancers.     

Radiofrequente ablatiebehandeling van de oudere patiënte met een mammacarcinoom

Breast cancer is an important health care problem, especially in the increasing elderly generation. Treatment of these fragile patients is a challenge for the clinician. Undertreatment has been linked to a higher percentage of recurrence and cancer related morbidity, while overtreatment leads to treatment related morbidity and mortality. Minimally invasive techniques do offer new opportunities for patients, who are no candidates for conventional surgery. The tumor lesion is treated locally and selective with minimal damage to surrounding tissue, yielding an adequate local tumor control. Radio frequency ablation technique seems an effective and safe method for treatment of the elderly patient with small (< 3 cm) breast cancer.     

Oxygen transport in the cerebral cortex of rats breathing a hyperoxic gas mixture]

Oxygen dissolved in the arterial blood plasma at a high pressure was shown to pass into the brain tissue from the finest arterioles. Therefore only a thin layer of the tissue immediately adjacent to these vessels is affected by the increased oxygen tension pO2. Permeability of the arteriole walls for oxygen protects the neurones against the high pO2. A special physiological feature of the oxygen transport during normobaric hyperoxia in the brain tissue involves very "steep" gradients of the pO2 in tissues and of the transferring the oxygen fraction from arterioles to venules through the tissues. The findings allow to compare distribution of the pO2 over the whole brain vessel network with that during inhalation of air or pure oxygen.     

Tissue-, age-, and stage-specific patterns of protein synthesis during the development of Drosophila melanogaster.

Patterns of polypeptide synthesis in the imaginal and larval tissues of wild-type larvae of different ages have been examined using a one-dimensional polyacrylamide gel electrophoresis and autofluorography. Tissue-, age, and stage-specific patterns of polypeptide bands have been observed and characterized. There are one or two bands unique to larval tissues and 13 bands unique to imaginal discs and brain. Each tissue has its own characteristic band pattern. The 160-hr wing disc contains all 13 disc and brain-specific bands plus another eight unique bands. The 160-hr leg disc contains 11 disc- and brain-specific bands plus another six unique bands. The 160-hr eye-antennal disc contains eight disc- and brain-specific bands. There are five bands common to all imaginal tissues at all times but not found in any of the larval tissues. There appears to be a much greater difference between the same tissue at different ages than there is between different tissues at the same age. There is also a marked change in the total body proteins extracted from different developmental stages. Eight (11%) of the bands detected appear to be common to all tissues at all times, while 13 bands (18%) appear to be specific to pairs of imaginal discs. In addition, there are major differences in the patterns observed between pulsed and pulse-chased animals. These results are discussed in relation to the concept of differential gene activity.     

The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications

The emergence of electron tomography as a tool for three dimensional structure determination of cells and tissues has brought its own challenges for the preparation of thick sections. High pressure freezing in combination with freeze substitution provides the best method for obtaining the largest volume of well-preserved tissue. However, for deeply embedded, heterogeneous, labile tissues needing careful dissection, such as brain, the damage due to anoxia and excision before cryofixation is significant. We previously demonstrated that chemical fixation prior to high pressure freezing preserves fragile tissues and produces superior tomographic reconstructions compared to equivalent tissue preserved by chemical fixation alone. Here, we provide further characterization of the technique, comparing the ultrastructure of Flock House Virus infected DL1 insect cells that were (1) high pressure frozen without fixation, (2) high pressure frozen following fixation, and (3) conventionally prepared with aldehyde fixatives. Aldehyde fixation prior to freezing produces ultrastructural preservation superior to that obtained through chemical fixation alone that is close to that obtained when cells are fast frozen without fixation. We demonstrate using a variety of nervous system tissues, including neurons that were injected with a fluorescent dye and then photooxidized, that this technique provides excellent preservation compared to chemical fixation alone and can be extended to selectively stained material where cryofixation is impractical.     

Migration-Directing Liquid Properties of Embryonic Amphibian Tissues

Deep ectoderm, mesoderm and endoderm excised from gastrulatingamphibian embryos spontaneously undergo liquid-like movementsin organ culture. Cell populations of these tissues on nonadhesivesubstrata will round up into spheres, spread over one anotherand segregate (sort out) from one another just as immiscibleliquid droplets do. In ordinary liquids, movements like theseare controlled by surface tensions; perhaps surface tensionsalso control the similar movements of liquid-like tissues. Onenecessary condition for tissue surface tension analysis is thatthe tissue must be able (just as ordinary liquids are able)to spontaneously relax internal stretching forces (shear stresses).When cellular aggregates of the germ layers were deformed bygentle compression between parallel glass plates, cells withinthe aggregates were initially stretched. However, the cellssoon returned to their original undistorted shapes. Thus, cellstretching forces were gradually relaxed by cell rearrangements.The in vitro spreading movements of the deep germ layers implythat the surface tension of ectoderm should be greater thanthe surface tension of mesoderm which should be greater thanthe surface tension of endoderm. Quantitative measurements oftissue surface tensions made by parallel plate compression confirmprecisely that relationship. Furthermore, the surface tensionsof these tissues remain constant regardless of the amount ofaggregate flattening—another necessary condition for validsurface tension measurements. These results demonstrate thatamphibian deep germ layers possess fundamental liquid propertieswhich are sufficient to direct their liquid-like rearrangementsin organ culture. Furthermore, I also report that one of theseproperties, surface tension, displays a preliminary correlationwith density of cell surface charge (assessed by electrophoreticmobility) and with the onset of in vivo mesodermal involution.