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Authors: Mª José Gómez Torres, Javier Rodríguez santero, Javier Gil Flores
Proper citation: Mª José Gómez Torres, Javier Rodríguez santero, Javier Gil Flores 2019. CUESTIONARIO PARA PARTICIPANTES EN PROGRAMAS DE INTERVENCIÓN SOCIOLABORAL. protocols.io dx.doi.org/10.17504/protocols.io.uxdexi6 Copy
Authors: Melissa Pontes Pereira, María Cristina Vidal Pessolani
Summary: Goal:Immunohistochemistry (or IHC) is a method that allows demonstrating the presence and location of proteins in tissue sections.Day 1:Thaw the slides about 15 minutes.Wash the slides 1 x 15 minutes in PBS with gentle agitation.Wipe away excess liquid around the section on the glass slide with tissue paper.Encircle the tissue section or draw lines on both sides of the section and let dry (10-15 seconds). The PAP PEN is designed to provide a water repellant barrier when a circle is drawn around a specimen such as tissue sections or cells. The barrier of the PAP PEN retains antisera within the defined area and ensures that only the amount of antibody needed for sufficient reaction is used.Block in 5% normal serum with 2% BSA in PBS plus 0.3% Triton X-100 (block solution) for 4 hours at room temperature.Drain slides for a few seconds (do not rinse)Wash the slides 1 x 15 minutes in PBS with gentle agitation.Wipe away excess liquid around the section on the glass slide with tissue paper.Apply primary antibody diluted in block solutionIncubate overnight at 4°C Day 2Drain slides for a few seconds (do not rinse)Wash the slides 2 x 15 minutes in PBS 0.05% Tween 20 with gentle agitationWash the slides 1 x 15 minutes in PBS with gentle agitationWipe away excess liquid around the section on the glass slide with tissue paper.Apply fluorophore-conjugated secondary antibody to the slide diluted to the concentration recommended by the manufacturer in block solution, and incubate for 2 hours at room temperature. This step should be done in the dark to avoid photobleaching.Wash the slides 2 x 15 minutes in PBS 0.05% Tween 20 with gentle agitationWash the slides 1 x 15 minutes in PBS with gentle agitationWipe away excess liquid around the section on the glass slide with tissue paper.Apply DAPI 1:100 in PBS 1X for 5 min at room temperature.Rinse in running tap distilled waterCoverslip with mounting medium.Day 2Drain slides for a few seconds (do not rinse)Wash the slides 2 x 15 minutes in PBS 0.05% Tween 20 with gentle agitationWash the slides 1 x 15 minutes in PBS with gentle agitationWipe away excess liquid around the section on the glass slide with tissue paper.Apply fluorophore-conjugated secondary antibody to the slide diluted to the concentration recommended by the manufacturer in block solution, and incubate for 2 hours at room temperature. This step should be done in the dark to avoid photobleaching.Wash the slides 2 x 15 minutes in PBS 0.05% Tween 20 with gentle agitationWash the slides 1 x 15 minutes in PBS with gentle agitationWipe away excess liquid around the section on the glass slide with tissue paper.Apply DAPI 1:100 in PBS 1X for 5 min at room temperature.Rinse in running tap distilled waterCoverslip with mounting medium.Attention!! All incubations should be carried out in a humidified chamber to avoid drying of the tissue.ControlsTo estimate the contribution of the non-specific interaction and Fc receptor binding, staining protocols using an antibody directed to an irrelevant antigen (for example, BrdU) having the same isotype as the antibody of interest may be analyzed in parallel with the antibody of interest. The antibody directed to the irrelevant antigen is known as the isotype control. For whole serum antibodies, use normal serum from an unimmunized animal of the same species as the primary antibody. If an isotype control is not available, a negative antibody control is recommended. Simply replace the primary antibody with antibody diluent. A positive tissue control is strongly recommended to ensure that the antibody is performing as expected. Depending on the experiment, it may also be useful to include a negative tissue control: a tissue in which the protein of interest is not expected to be found.Referenceswww.abcam.com/technical
Proper citation: Melissa Pontes Pereira, María Cristina Vidal Pessolani 2018. Immunohistochemistry (or IHC) Protocol for frozen sections. protocols.io dx.doi.org/10.17504/protocols.io.pkmdku6 Copy
Authors: Manuel Liebeke
Summary: TAHS was synthetized according to the protocol previously described with minor modifications. TAHS is used as derivatization reagent for amino acids. The purpose is to generate enough TAHS for on-tissue derivatisation to enhance signals during MALDI-MSI of amino acids from tissue sections.
Proper citation: Manuel Liebeke 2019. Synthesis of derivatization reagent TAHS (N,N,N-trimethylamonioanilyl N-hydroxysuccinimidyl carbamate iodide). protocols.io dx.doi.org/10.17504/protocols.io.j8ycrxw Copy
Authors: Lgc Biosearch Technologies
Summary: A set of Stellaris FISH Probes comprises up to 48 singly labeled oligonucleotides designed to selectively bind to targeted transcripts. Stellaris FISH Probes bound to target RNA produce fluorescent signals that permit detection of single RNA molecules as diffraction-limited spots by conventional fluorescence microscopy. Please see the Biosearch
Proper citation: Lgc Biosearch Technologies 2016. Stellaris® RNA FISH Protocol for Frozen Tissue. protocols.io dx.doi.org/10.17504/protocols.io.cdqs5v Copy
Authors: Miriam Heinen
Summary: This staining was performed to detect fetuin-A in activated microglia. CD68 is a marker, that stains activated microglia, macrophages and monocytes. Fetuin-A and microglia were detected in paraffin sections (1 μm thickness) of formalin-fixed human brain tissue. CD68 was stained using a monoclonal mouse-anti-human antibody (Dako Cat# M0814, RRID:AB_2314148, clone KP1, dilution 1:50) and a polyclonal goat-anti-mouse Alexa Fluor® 488 conjugated secondary antibody (Thermo Fisher Scientific Cat# A-11029, RRID:AB_2534088, dilution 1:300). Fetuin-A was detected by using a monoclonal IgG2a mouse-anti-human antibody (clone MAHS-1, dilution 1.0 µg/mL), raised against purified human fetuin-A in our laboratories. Antibody binding was detected by tyramide signal amplification using a secondary biotinylated polyclonal goat-anti-mouse antibody (Dako Cat# E0433, RRID:AB_2687905, dilution 1:300) and a Tyramide Signal Amplification Kit (Life Technologies, Carlsbad, USA, T-20933). To minimize lipofuscin autofluorescence, sections were counterstained with Sudan Black (Sigma-Aldrich, Munich, Germany, 199664, dilution 0.3% in 70% ethanol, 5 minutes). Nuclei were stained with DAPI (Sigma-Aldrich, Munich, Germany D9542, dilution 0.25 µg/ml, 5 minutes). Sections were mounted with Immumount (Thermo Scientific, Waltham, USA, 9990402) and stored at 8°C in the dark.
Proper citation: Miriam Heinen 2018. Immunofluorescence staining protocol for co-staining of fetuin-A and CD68 in older human autopsy tissue. protocols.io dx.doi.org/10.17504/protocols.io.syfeftn Copy
Authors: Frank Aylward
Summary: This is an example of a simple short read mapping analysis that could be used as part of a transcriptomics or RNA-seq workflow. Code is intended for use on an Ubuntu 16.04 LTS OS, but it may work on other Unix or Unix-like systems.This tutorial uses data from the following very nice paper from the Hatfull group:Dedrick, Mavrich, Ng, and Hatfull, Expression and evolutionary patterns of mycobacteriophage D29 and its temperate close relatives. BMC Microbiology, 2017. https://doi.org/10.1186/s12866-017-1131-2The tools that are used include:SRA toolkit: https://www.ncbi.nlm.nih.gov/sra/docs/toolkitsoft/bowtie2: http://bowtie-bio.sourceforge.net/bowtie2/index.shtmlsamtools: http://samtools.sourceforge.net/BEDOPS: https://bedops.readthedocs.io/en/latest/content/reference/file-management/conversion/bam2bed.htmlBEDtools: https://bedtools.readthedocs.io/en/latest/Make sure that these tools are installed before starting the tutorial. On a Ubuntu OS you should be able to install most with "sudo apt install", but you may wish to use a package manager such as Anaconda or Miniconda as well. Throughout the tutorial some notes may be made about particular versions of tools. For example, note that the version of SRA toolkit that is installed with apt install often leads to downstream errors, it may be desirable to install this tool with Miniconda instead. Miniconda bash installers can be found here: https://conda.io/miniconda.html
Proper citation: Frank Aylward 2018. Introduction to read mapping for transcriptomics. protocols.io dx.doi.org/10.17504/protocols.io.qhedt3e Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the process to obtain electrophysiological recordings and cellular contents from neurons in postnatal mouse and/or human brain slices.Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Proper citation: Allen Institute for Brain Science 2020. Patch-Seq Recording and Extraction. protocols.io dx.doi.org/10.17504/protocols.io.bdpei5je Copy
Authors: Sebastiaan Kuiper
Group: iGEM Wageningen 2019
Summary: To observe the potential of defense mechanisms of either native or synthetic systems in Escherichia coli (and more) when incubated with a bacteriophage stock.
Proper citation: Sebastiaan Kuiper 2019. Growth curve analysis. protocols.io dx.doi.org/10.17504/protocols.io.77jhrkn Copy
Authors: Sam Li
Group: BioLegend
Summary: BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with pre-diluted MojoSort™ reagents and using the columns as indicated by the manufacturer.Note: Due to the properties of our beads, it may be possible to use far fewer beads that with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:3 to 1:20 for the Nanobeads can be used. Please contact BioLegend Technical Service ([email protected]) if further assistance is needed.
Proper citation: Sam Li 2019. MojoSort™ Human anti-PE Nanobeads Column Protocol. protocols.io dx.doi.org/10.17504/protocols.io.7aghibw Copy
Authors: Nam JH, Kim DH, Yoo JH, Hwang JH, Chang JD
Summary: Background Elderly patients undergoing hip fracture surgery (HFS) are at increased risk of postoperative venous thromboembolism (VTE). Therefore, combined postoperative mechanical and chemical thromboprophylaxis has been routinely performed after HFS in these patients. This retrospective case-control study was conducted to evaluate the additional effectiveness of preoperative mechanical thromboprophylaxis on the prevention of VTE following HFS in elderly patients.Methods Of 539 consecutive patients aged 70 years or older undergoing HFS, 404 (control group) did not receive preoperative mechanical thromboprophylaxis while 135 (study group) received mechanical thromboprophylaxis using an intermittent pneumatic compression device and graduated compression stockings from the time of admission until surgery. All patients received combined postoperative mechanical and chemical thromboprophylaxis following HFS in accordance with the same protocol. The incidence of symptomatic VTE confirmed by clinical symptoms and 3-dimensional CT angiography within one month of surgery was investigated in both groups.Results American Society of Anesthesiologists grade was higher (p=0.016) in the study group and more patients in this group had concomitant cardiovascular and neurologic diseases (p=0.005 and p=0.009, respectively). Meanwhile, more patients in the study group had received anticoagulant medication preinjury owing to comorbidities (39% vs 28%, p=0.025). The overall incidences of symptomatic deep vein thrombosis (DVT) and pulmonary embolism (PE) were 7.4% and 3.7% in the control group and 2.2% and 1.5% in the study group, respectively. On multiple logistic regression, symptomatic DVT significantly reduced in the study group (OR 0.28, p=0.042), meanwhile there was no significant difference in the incidence of symptomatic PE between the two groups (p=0.223).Conclusions Preoperative mechanical thromboprophylaxis may confer an additional benefit by preventing postoperative VTE without adding more risk of perioperative bleeding in elderly patients with hip fracture.
Proper citation: Nam JH, Kim DH, Yoo JH, Hwang JH, Chang JD 2017. Preoperative mechanical prophylaxis in elderly patients with hip fracture. protocols.io dx.doi.org/10.17504/protocols.io.jfscjne Copy
Authors: Bruce Mcdonald
Group: Zymoseptoria community protocols (STBnet)
Summary: Bruce McDonald 1990 modified from Murray and Thompson
Proper citation: Bruce Mcdonald 2018. Total DNA "Midi" CTAB prep. for Zymoseptoria tritici. protocols.io dx.doi.org/10.17504/protocols.io.s2wegfe Copy
Authors: Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]
Summary: This IgY purification technology can be used to purified IgY from the egg yolk of any bird as ducks, bantam hens, ostrich, chicken, pigeon, pheasant, quail, guinea hen, cattle egret and goose.
Proper citation: Angel Justiz-Vaillant 2020. Purification of the any avian IgY using chloroform, trichloroacetic acid and affinity purification using HiTrap™ Columns.. protocols.io dx.doi.org/10.17504/protocols.io.bjnskmee Copy
Authors: Etienne Laliberté, Raymond Soffer
Group: Canadian Airborne Biodiversity Observatory
Summary: Here we describe the standardised protocol used by the Canadian Airborne Biodiversity Observatory (CABO) to measure leaf spectral reflectance and transmittance, using the Spectra Vista Corporation (SVC) DC-R/T Integrating Sphere fitted to a portable full-range SVC HR-1024i field spectroradiometer. This standard version of our protocol describes the common case where an individual leaf is large enough to entirely cover the reflectance or transmission port of the integrating sphere. Briefly, six mature, healthy-looking and sunlit leaves from a canopy plant are selected for measurements of adaxial reflectance and transmittance. Leaf scans are referenced to a calibrated Spectralon® disk and corrected for stray light. Our leaf spectroscopy protocol builds from that of the Carnegie Airborne Observatory.
Proper citation: Etienne Laliberté, Raymond Soffer 2018. Measuring spectral reflectance and transmittance (350-2500 nm) of large leaves using the Spectra Vista Corporation (SVC) DC-R/T Integrating Sphere. protocols.io dx.doi.org/10.17504/protocols.io.pzbdp2n Copy
Authors: Elena Casacuberta, Aleksandra Kozyczkowska, Sebastian Najle
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab, Emerging Organisms for Biology
Summary: This is a protocol for transient transfection of the Icthyosporean Abeoforma whisleri. This protocol has an efficiency of 1-2% calculated from fluorescent positive cell from Flow Cytometry data, using the amount of cells and DNA described in the protocol. Increasing DNA from above 1 microgram/microliter does not increase transfection.
Proper citation: Elena Casacuberta, Aleksandra Kozyczkowska, Sebastian Najle 2019. Abeoforma whisleri transient transfection protocol. protocols.io dx.doi.org/10.17504/protocols.io.zexf3fn Copy
Authors: Bing Yang
Proper citation: Bing Yang 2019. DNA Extraction from Urine. protocols.io dx.doi.org/10.17504/protocols.io.3awgife Copy
Authors: Remco Stam
Proper citation: Remco Stam 2016. Cultivation / Spore production for Fusarium spp. protocols.io dx.doi.org/10.17504/protocols.io.fmtbk6n Copy
Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: This protocol was used to dissociate adult (8-10 wk) mouse lung tissue. The entire procedure is carried out on ice (to reduce artifact gene expression changes) and takes about half an hour. The yield was 16,240 non-RBC/mg tissue with 87% viability.
Proper citation: Andrew Potter 2018. Adult mouse lung cell dissociation (on ice). protocols.io dx.doi.org/10.17504/protocols.io.q6bdzan Copy
Authors: Benjamin Bolduc
Group: Sullivan Lab
Summary: Assembling actual reads from the Ocean Sampling Day (2014) using SPAdes, an assembler implemented in Cyverse.
Proper citation: Benjamin Bolduc 2016. Assembling Viral Metagenomic Data with SPAdes (Cyverse). protocols.io dx.doi.org/10.17504/protocols.io.evzbe76 Copy
Authors: LeinU01 BRAIN Grant
Group: BICCN, Allen Institute for Brain Science
Summary: Isolation of nuclei from frozen adult human brain tissue or thawed and microdissected brain tissue sections for FPCR and/or RNA-seq analysis.
Proper citation: LeinU01 BRAIN Grant 2019. Isolation of Nuelci from Adult_Human_Brain_Tissue. protocols.io dx.doi.org/10.17504/protocols.io.y6pfzdn Copy
Authors: Teesha Luehr
Group: Leonard Foster's Lab
Proper citation: Teesha Luehr 2019. STAGE (STop And Go Extraction) C18 tips for Desalting and Clean Up. protocols.io dx.doi.org/10.17504/protocols.io.y9pfz5n Copy
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