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1.
A practical approach to routine immunostaining of paraffin sections in the microwave oven 总被引:1,自引:0,他引:1
M. E. Boon F. C. J. Hendrikse P. G. Kok P. Bolhuis L. P. Kok 《The Histochemical journal》1990,22(6-7):347-352
Summary The Streptavidin-Biotin Complex (SABC) Immunostaining method can be carried out by performing the rinsing and blocking steps and in addition the incubations with the primary and the secondary antibody sera in the microwave oven. Irradiation of the Streptavidin-Biotin Complex reduces stain activity due to destruction of horseradish peroxidase (HRP) (Boon & Kok, 1988). Therefore, we decided not to perform this step in the microwave oven. The microwave incubations can be performed using antisera dilutions of 1:1000 (instead of 1:50) in tissue fixed with Kryofix, allowing staining in staining racks. This keeps hands-on time low and simplifies the microwave steps. It is very difficult to obtain reproducible results in the microwave oven using droplet incubations due to problems with hot spots and antenna effects. These problems are avoided when cuvettes are used and air is blown through the solutions during microwave incubations. With effective temperature control the method is highly reproducible, and takes at least 100 min less than the conventional SABC method. It is, in particular, attractive when large series of slides must be routinely immunostained and when reproducible results are desired. 相似文献
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A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques 总被引:2,自引:2,他引:0
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HA Lester ME Krouse MM Nass NH Wassermann BF Erlanger 《The Journal of general physiology》1980,75(2):207-232
After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules. 相似文献
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Helene Hartwig Carlos Silvestre-Roig Jeffrey Hendrikse Linda Beckers Nicole Paulin Kim Van der Heiden Quinte Braster Maik Drechsler Mat J. Daemen Esther Lutgens Oliver Soehnlein 《PloS one》2015,10(10)
Atherosclerosis-associated diseases are the main cause of mortality and morbidity in western societies. The progression of atherosclerosis is a dynamic process evolving from early to advanced lesions that may become rupture-prone vulnerable plaques. Acute coronary syndromes are the clinical manifestation of life-threatening thrombotic events associated with high-risk vulnerable plaques. Hyperlipidemic mouse models have been extensively used in studying the mechanisms controlling initiation and progression of atherosclerosis. However, the understanding of mechanisms leading to atherosclerotic plaque destabilization has been hampered by the lack of proper animal models mimicking this process. Although various mouse models generate atherosclerotic plaques with histological features of human advanced lesions, a consensus model to study atherosclerotic plaque destabilization is still lacking. Hence, we studied the degree and features of plaque vulnerability in different mouse models of atherosclerotic plaque destabilization and find that the model based on the placement of a shear stress modifier in combination with hypercholesterolemia represent with high incidence the most human like lesions compared to the other models. 相似文献
4.
Hunter A Hendrikse A Renan M Abratt R 《Apoptosis : an international journal on programmed cell death》2006,11(10):1727-1735
Cytotoxic anti-cancer agents induce apoptosis in tumor and normal tissues. Therefore, it is important to investigate which
factors determine these apoptotic processes and hence their likely impact on therapeutic gain. Radiation-induced apoptosis
in tumors may be inhibited due to mutations of apoptotic elements or to tumor microenvironmental conditions arising from vascular
insufficiency. Tumors typically contain regions of hypoxia, low glucose and acidosis. Hypoxic cells compromise treatment partly
because of reduced fixation of damage during radiotherapy and partly because they promote a more malignant phenotype. There
is also evidence that hypoxia may inhibit apoptosis. For some cell types, concurrent hypoxia may modulate radiation-induced
apoptosis while, for others, post-irradiation hypoxia may be required. This may reflect the activity of different apoptotic
pathways. Pathways involving mitochondrial components as well as regulation of SAPK and Fas have been implicated. In addition,
several key stages in apoptosis are sensitive to depletion of cellular energy reserves, which results from hypoxia and low
glucose conditions. There is also evidence that low pH in tumors can interfere with radiation-induced apoptosis, partly through
cell cycle arrest and other undefined mechanisms. Conclusions: Hypoxia, low glucose and acidosis influence radiation-induced apoptosis and thus may be detrimental to radiotherapy. 相似文献
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SUMMARY Research conducted under the label of evolutionary developmental biology has tended to revolve around a few central issues such as modularity, integration, and canalization. Yet, as the field has grown, it has become increasingly difficult to define in terms of its central question and relation to broader evolutionary concerns. We argue that these central issues of evo-devo gain their currency from connections to a central question that defines the field, and we propose that this central question is about the nature of evolvability. However, not all research currently carried out under the label of "evo-devo" speaks to this focal concern. The aim of this article is therefore to argue for a precise formulation of evolutionary developmental biology's core question. 相似文献
6.
JM Devaney S Wang P Furbert-Harris V Apprey M Ittmann B-D Wang J Olender NH Lee B Kwabi-Addo 《Epigenetics》2015,10(4):319-328
Increasing evidence suggests that aberrant DNA methylation changes may contribute to prostate cancer (PCa) ethnic disparity. To comprehensively identify DNA methylation alterations in PCa disparity, we used the Illumina 450K methylation platform to interrogate the methylation status of 485,577 CpG sites focusing on gene-associated regions of the human genome. Genomic DNA from African-American (AA; 7 normal and 3 cancers) and Caucasian (Cau; 8 normal and 3 cancers) was used in the analysis. Hierarchical clustering analysis identified probe-sets unique to AA and Cau samples, as well as common to both. We selected 25 promoter-associated novel CpG sites most differentially methylated by race (fold change > 1.5-fold; adjusted P < 0.05) and compared the β-value of these sites provided by the Illumina, Inc. array with quantitative methylation obtained by pyrosequencing in 7 prostate cell lines. We found very good concordance of the methylation levels between β-value and pyrosequencing. Gene expression analysis using qRT-PCR in a subset of 8 genes after treatment with 5-aza-2′-deoxycytidine and/or trichostatin showed up-regulation of gene expression in PCa cells. Quantitative analysis of 4 genes, SNRPN, SHANK2, MST1R, and ABCG5, in matched normal and PCa tissues derived from AA and Cau PCa patients demonstrated differential promoter methylation and concomitant differences in mRNA expression in prostate tissues from AA vs. Cau. Regression analysis in normal and PCa tissues as a function of race showed significantly higher methylation prevalence for SNRPN (P = 0.012), MST1R (P = 0.038), and ABCG5 (P < 0.0002) for AA vs. Cau samples. We selected the ABCG5 and SNRPN genes and verified their biological functions by Western blot analysis and siRNA gene knockout effects on cell proliferation and invasion in 4 PCa cell lines (2 AA and 2 Cau patients-derived lines). Knockdown of either ABCG5 or SNRPN resulted in a significant decrease in both invasion and proliferation in Cau PCa cell lines but we did not observe these remarkable loss-of-function effects in AA PCa cell lines. Our study demonstrates how differential genome-wide DNA methylation levels influence gene expression and biological functions in AA and Cau PCa. 相似文献
7.
Multidrug resistance (MDR) is characterized by the occurrence of cross-resistance to a broad range of structurally and functionally unrelated drugs. Several mechanisms are involved in MDR. One of the most well-known mechanisms is the overexpression of P-glycoprotein (P-gp), encoded by the MDR1 gene in humans and by the mdr1a and mdr1b genes in rodents. P-gp is extensively expressed in the human body, e.g., in the blood-brain barrier and also in solid tumor tissue. Overexpression of P-gp on tumor membranes might result in MDR of human tumors. To circumvent this resistant phenotype, several P-gp modulators such as cyclosporin A (CsA) are available. Competition between P-gp drugs and modulators results in decreased transport of the drug out of tumor tissue and an increased cellular level of these drugs. For effective clinical treatment it is important to have knowledge about P-gp functionality in tumors. Therefore, we have developed a method to measure the P-gp functionality in vivo with PET and [(11)C]verapamil as a positron-emitting P-gp substrate. The results obtained in rodents and in cancer patients are described in this article. 相似文献
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Anita A. Harteveld Laurens J. L. De Cocker Nikki Dieleman Anja G. van der Kolk Jaco J. M. Zwanenburg Pierre A. Robe Peter R. Luijten Jeroen Hendrikse 《PloS one》2015,10(3)