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Rehydration capacities and rates for various porcine tissues after dehydration.
PUBLISHED: 01-01-2013
The biphasic effects of liquid on tissue biomechanics are well known in cartilage and vocal folds, yet not extensively in other tissue types. Past studies have shown that tissue dehydration significantly impacts biomechanical properties and that rehydration can restore these properties in certain tissue types. However, these studies failed to consider how temporal exposure to dehydrating or rehydrating agents may alter tissue rehydration capacity, as overexposure to dehydration may permanently prevent rehydration to the initial liquid volume. Select porcine tissues were dehydrated until they reached between 100% and 40% of their initial mass. Each sample was allowed to rehydrate for 5 hours in a 0.9% saline solution, and the percent change between the initial and rehydrated mass values was calculated. Spearman correlation tests indicated a greater loss in mass despite rehydration when tissues were previously exposed to greater levels of dehydration. Additionally, Pearson correlation tests indicated the total liquid mass of samples after complete rehydration decreased when previously exposed to higher levels of dehydration. Rehydration rates were found by dehydrating tissues to 40% of their initial mass followed by rehydration in a 0.9% saline solution for 60 minutes, with mass measurements occurring in 15 minute intervals. All tissues rehydrated nonlinearly, most increasing significantly in mass up to 30 minutes after initial soaking. This study suggests the ability for tissues to rehydrate is dependent on the level of initial dehydration exposure. In vitro rehydration experiments therefore require controlled dosage and temporal exposure to dehydrating and rehydrating agents to avoid incomplete rehydration, and caution should be taken when combining different tissue types in models of hydration.
Authors: Billie Velapatiño, James E. A. Zlosnik, Trevor J. Hird, David P. Speert.
Published: 10-15-2013
The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.
24 Related JoVE Articles!
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DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses
Authors: Larissa A. Pikor, Katey S. S. Enfield, Heryet Cameron, Wan L. Lam.
Institutions: BC Cancer Research Centre, University of British Columbia - UBC, BC Cancer Agency, University of British Columbia - UBC.
Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20°C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.
Genetics, Issue 49, DNA extraction, paraffin embedded tissue, phenol:chloroform extraction, genetic analysis, epigenetic analysis
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Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry
Authors: Peter Nemes, Akos Vertes.
Institutions: George Washington University.
Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 μm wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 μm and ~40 μm, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.
Molecular Biology, Issue 43, imaging mass spectrometry, ambient mass spectrometry, direct analysis, tissue, biofilm
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MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues
Authors: Alison J. Kriegel, Mingyu Liang.
Institutions: Medical College of Wisconsin.
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Authors: Nadine Dyballa, Sabine Metzger.
Institutions: University Duesseldorf.
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RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis
Authors: Samuel M. Peterson, Jennifer L. Freeman.
Institutions: Purdue University.
Many important and complex laboratory procedures require an input of high quality, intact RNA. A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications. It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample. Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated. The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.
Developmental Biology, Issue 30, zebrafish, RNA, cDNA, expression, microarray, gene
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Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes
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Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion
Authors: Nathaniel T. Remlinger, Peter D. Wearden, Thomas W. Gilbert.
Institutions: McGowan Institute for Regenerative Medicine, University of Pittsburgh, Children's Hospital of Pittsburgh of UPMC, University of Pittsburgh.
Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.
Bioengineering, Issue 70, Tissue Engineering, Biomedical Engineering, Cellular Biology, Medicine, Cardiology, Extracellular matrix, decellularization, animal model, porcine, cardiac, heart tissue
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Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue
Authors: Molly J. McDonough, Chelsea E. Allen, Ng-Kwet-Leok A. Ng-Sui-Hing, Brian A. Rabe, Brittany B. Lewis, Margaret S. Saha.
Institutions: College of William and Mary.
Developmental Biology, Issue 70, Neuroscience, Cellular Biology, Surgery, Anatomy, Physiology, Ophthalmology, retina, primary cell culture, dissection, confocal microscopy, calcium imaging, fluorescent in situ hybridization, FISH, Xenopus laevis, animal model
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Skeletal Muscle Gender Dimorphism from Proteomics
Authors: Kalina Dimova, Lauren Ann Metskas, Mohini Kulp, Stylianos P. Scordilis.
Institutions: Smith College, Yale University, Smith College, Smith College.
Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p < 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).
Medicine, Issue 58, skeletal muscle, gender, 2-D gel electrophoresis, HPLC/MS, mouse
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Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves
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Optimized PCR-based Detection of Mycoplasma
Authors: Paige L. Dobrovolny, Dan Bess.
Institutions: Sigma-Aldrich.
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Institutions: Uppsala University, Swedish University of Agricultural Sciences.
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Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue
Authors: Jacek R. Wiśniewski.
Institutions: Max Planck Institute of Biochemistry.
Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.
Chemistry, Issue 79, Clinical Chemistry Tests, Proteomics, Proteomics, Proteomics, analytical chemistry, Formalin fixed and paraffin embedded (FFPE), sample preparation, proteomics, filter aided sample preparation (FASP), clinical proteomics; microdissection, SAX-fractionation
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Authors: Víctor A. Lórenz-Fonfría, Joachim Heberle.
Institutions: Freie Universität Berlin.
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Authors: Johannes Felix Buyel, Rainer Fischer.
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
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Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Institutions: University of Maine.
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Institutions: BioSentinel Inc., Madison, WI.
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Authors: Lawren Sack, Christine Scoffoni.
Institutions: University of California, Los Angeles .
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