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Authors: Ghazi Alsbeih, Krishna Mishra, Maha Alrashd, Subramanian M. Pulicat, Najla Al-Harbi, Sara Bin Judia, Belal Moftah
Proper citation: Ghazi Alsbeih, Krishna Mishra, Maha Alrashd, Subramanian M. Pulicat, Najla Al-Harbi, Sara Bin Judia, Belal Moftah 2019. Flow Cytometry-Based Gamma-H2AX Assay for Radiation Biodosimetry. protocols.io dx.doi.org/10.17504/protocols.io.5gzg3x6 Copy
Authors: Alexander Lazutkin
Group: Brain Stem Cell Lab
Summary: Labeling of the replicating DNA with synthetic thymidine analogs is commonly used for marking the dividing cells. However, until now this method has only been applied to histological sections. A growing number of current approaches for three-dimensional visualization of large tissue samples requires detection of dividing cells within whole organs. Here we describe a method for labeling dividing cells with 5-ethynyl-2'-deoxyuridine (EdU) and their further detection in whole brain structures (for example, hippocampus) using the Cu (I) -catalyzed [3 + 2] cycloaddition reaction (so-called click-reaction). The presented method can be used for brain neurogenesis studies as well as for whole-mount staining of any preparations in which the terminal ethynyl group has been introduced.• New click histochemistry method based on Cu (I) -catalyzed [3 + 2] cycloaddition reaction allows whole-mount staining of brain structures and other tissues.• Our whole-mount click histochemistry method allows to visualize dividing cells in 3D and can be used in neurogenesis studies, i.e. for birthdating dividing early progenitors and further tracking of proliferation, survival, migration, differentiation, and fate of their progeny.• Our whole-mount click histochemistry staining demonstrates high staining specificity, high signal intensity, and low background levels in young and adult mouse brain tissue.
Proper citation: Alexander Lazutkin 2019. Click histochemistry for whole-mount staining of brain structures. protocols.io dx.doi.org/10.17504/protocols.io.8rqhv5w Copy
Authors: Kerry Wiles
Group: Laboratory of Systems Pharmacology, NCIHTAN
Summary: Tissues are procured from a variety of different anatomic sites, each with their own unique qualities to be considered during collection. Anatomic site-specific standard operating procedures (SOPs) are required to address these unique qualities and assist with investigator-specific requests. This protocol for the collection of normal (NL) colon applies for normal adjacent tissue (NAT) in cancerous colons and also for histologically normal diverticulitis colon tissue.
Proper citation: Kerry Wiles 2021. Tissue Procurement: Normal Colon . protocols.io dx.doi.org/10.17504/protocols.io.6y9hfz6 Copy
Authors: Manuela Contin
Summary: Levodopa oral test Administration of a subacutest dose of levodopa (100 mg) plus benserazide (25 mg) or carbidopa (25 mg) under standardized conditions.Levodopa kinetic–dynamic monitoringMeasurements of plasma levodopa concentrations and clinical effects by objective motor tests (alternate finger tapping test).
Proper citation: Manuela Contin 2020. Levodopa kinetic-dynamic test. protocols.io dx.doi.org/10.17504/protocols.io.bbq8imzw Copy
Authors: Peter Anderson
Summary: This location contains the GDSC data set with 145 oncogene mutation statuses and ~1200 chemical descriptors. For instructions how to run binary classification (to predict compound activity vs inactivity) or regression (to predict values of log IC50), see the README.classification.txt and README.regression.txt files, respectively.
Proper citation: Peter Anderson 2019. GDSC data set and random forest model scripts . protocols.io dx.doi.org/10.17504/protocols.io.3j9gkr6 Copy
Authors: Ian Mackay, Judy Northill
Summary: This is my preferred, previously published [Ref 1], rhinovirus (RV) and enterovirus (EV) genotyping assay when seeking to identify the genotype of a respiratory picornavirus detected in a clinical sample extract. It is employed after use of a screening real-time RT-PCR has identified a respiratory picornavirus.I have not confirmed that it can detect every single RV genotype but I do know that it detects many from each of the three RV species (Human rhinovirus A, Human rhinovirus B and Human rhinovirus C) as well as at least some Human enterovirus (EV) genotypes.The assay picks up EVs due to the shared genetic similarities in the 5'UTR target region. EVs can be discriminated using subgenomic sequencing (see VP42 typing assay protocol), or simply described as 'respiratory EVs' since there is no specific-specific vaccine or treatment available anyway. This is a robust primary subgenomic sequencing assay. It is more sensitive than any VP1 protocols because it targets more conserved primer target sites. It produces a more reliable typing result than does the 5'UTR region alone.
Proper citation: Ian Mackay, Judy Northill 2019. Respiratory picornavirus genotyping conventional nested RT-PCR ("Wisdom VP42 assay"). protocols.io dx.doi.org/10.17504/protocols.io.9tyh6pw Copy
Authors: Andrés Ramírez, Albane Ruaud, Fernan Federici, Peter von Dassow
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol has been optimized for electroporation of natural communities in coastal surface waters. As natural communities may vary in different places we recomend to use this protocol as a starting point and re-optimize according to the results. Guidelines are provided.
Proper citation: Andrés Ramírez, Albane Ruaud, Fernan Federici, Peter von Dassow 2017. Electroporation of natural communities in sea water. protocols.io dx.doi.org/10.17504/protocols.io.hpnb5me Copy
Authors: Sabine St-Jean, Mark Vellend
Group: Canadian Airborne Biodiversity Observatory
Summary: Here we describe the standardised protocol used by the Canadian Airborne Biodiversity Observatory (CABO) to survey open vegetation (i.e., vegetation without tree cover) at the Cowichan Garry Oak Preserve (British Columbia), Mer Bleue Bog (Ontario) and Parc national des Îles-de-Boucherville (Québec) sites. Surveys were conducted in 3x3m square plots, with each plot containing nine 1x1m subplots. Plot locations were selected in order to capture a range of environmental conditions of interest (e.g., distance from forest, soil types, or microtopography). All data were entered via the Fulcrum application, using the Plots, Subplots, and Vegetation Surveys: Herbs and Shrubs apps. For each plot, we first verified plot orientation (two edges of the square north-south, two east-west), measured geographic coordinates of the plot center and corners, and estimated slope angle and aspect. All plant species within the plot were identified. For each subplot, we made visual estimates of percent cover of all plant species present, as well as leaf litter and bare ground. If a drone was available, percent cover estimates were not made in the field; rather, overhead photographs taken with the drone were first annotated and later analyzed quantitatively using virtual point frames to obtain data on the abundance and distribution of plant species within the plots. The ground-based plant surveys were conducted in order to be paired with remotely-sensed aerial hyperspectral imagery.
Proper citation: Sabine St-Jean, Mark Vellend 2020. Open Vegetation Survey Protocol. protocols.io dx.doi.org/10.17504/protocols.io.3ebgjan Copy
Authors: Eric Carpenter
Group: GigaScience Press
Summary: Implemented by: Sarah Covshoff, Rowan Sage and Julian HibberdThis RNA isolation method is a multi-component method involving an initial extraction by hotacid phenol and then a purification and DNase treatment using the RNeasy Mini Kit by Qiagen.The method described below is a modification of a method described by van Tunen et al.3.This protocol is part of a collection of eighteen protocols used to isolate total RNA from plant tissue. (RNA Isolation from Plant Tissue Collection: https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6) 3 van Tunen, A.J. et al. Cloning of the two chalcone flavanone isomerase genes from Petunia hybrida: coordinate, light‐ regulated and differential expression of flavonoid genes. The EMBO Journal 7, 1257‐1263 (1988).
Proper citation: Eric Carpenter 2019. RNA Isolation from Plant Tissue Protocol 12: Hot Acid Phenol Method for Angiosperms. protocols.io dx.doi.org/10.17504/protocols.io.4rxgv7n Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Serum Urea/BUN levels are affected by alterations in systemic protein and nitrogen metabolism. Serum Urea/BUN levels are altered in kidney failure and renal complications of diabetes.
Proper citation: Jason Kim 2019. U Mass - Urea/BUN. protocols.io dx.doi.org/10.17504/protocols.io.x5qfq5w Copy
Authors: Delphine Hequet
Summary: Please see attached protocol
Proper citation: Delphine Hequet 2017. Prospective study evaluating the clinical impact of the Breast Cancer Intrinsic Subtype-ProsignaTM Test (Assay) in the management of early-stage breast cancers.. protocols.io dx.doi.org/10.17504/protocols.io.jrucm6w Copy
Authors: Qi Wang, Qiang Sun, Xiaoping Li, Zhefeng Wang, Haotian Zheng, Yanmei Ju, Ruijin Guo, Songlin Peng, Huijue Jia
Group: BGI, GIGA, GigaScience Press
Summary: Bone mass loss contributes to the risk of bone fracture in the elderly. Many factors including age, obesity, estrogen and diet, are associated with bone mass loss. Mice studies suggested that the gut microbiome might affect the bone mass by regulating the immune system, however there has been little evidence from human studies. Bone loss increases after menopause. Therefore, we have recruited 361 Chinese post-menopausal women to collect their fecal samples and metadata to conduct metagenome-wide association study (MWAS) to investigate the influence of the gut microbiome on bone health. Gut microbiome sequencing data were produced using BGISEQ500 sequencing, Bone mineral density (BMD) was calculated using Hologic dual energy X-ray machine, body mass index (BMI) and age were also recorded.This collected data allows exploration of the gut microbial diversity and their links to bone mass loss, as well as microbial markers for bone mineral density. In addition, these data are potentially useful in studying the role the gut microbiota might play in bone mass loss and in exploring the bone mass loss process.
Proper citation: Qi Wang, Qiang Sun, Xiaoping Li, Zhefeng Wang, Haotian Zheng, Yanmei Ju, Ruijin Guo, Songlin Peng, Huijue Jia 2021. Protocols for "Linking gut microbiome to bone mineral density: a shotgun metagenomic study of 361 eldly women". protocols.io dx.doi.org/10.17504/protocols.io.bq6imzce Copy
Authors: Ioannis Bellos
Summary: Autosomal dominant polycystic kidney disease (ADPKD) represents the most prevalent life-threatening monogenetic hereditary kidney disease. Hypertension occurs early in the course of the disease and is linked to progressive decline of glomerular filtration rate, as well as with higher cardiovascular morbidity. Experimental studies support that both polycystin 1 and 2 are expressed in the cilia of endothelial and vascular smooth muscle cells, serving as mechanoreceptors that sense shear stress. Loss of polycystins may lead to reduced nitric oxide production, promoting vasoconstriction, renal hypoxia and functional decline. Several observational studies have recently evaluated endothelial damage in ADPKD patients by using surrogate markers, although no firm consensus exists about endothelial dysfunction and arterial stiffness in early-stage patients with preserved renal function. Therefore, a meta-analysis is planned in order to accumulate current literature evidence in the field and compare makers of endothelial dysfunction and arterial stiffness among early-stage ADPKD patients with preserved renal fucntion and healthy controls.
Proper citation: Ioannis Bellos 2020. Markers of endothelial dysfunction and arterial stiffness in patients with early-stage autosomal dominant polycystic kidney disease: a meta-analysis. protocols.io dx.doi.org/10.17504/protocols.io.bcmfiu3n Copy
Authors: Tinsae Alemayehu
Summary: Background: Community nasal meningococcal carriage rates are high across Africa.Meningococcal infections are major causes of morbidity and mortality in the continent;especially among children and adolescents. This study aimed to determine the prevalence ofnasal carriage and antibiotic susceptibilities of meningococcal isolates from healthy Ethiopianchildren and adolescents.Method: A cross-sectional study was conducted in one of the sub-cities of AddisAbaba, Ethiopia. Nasal swabs were collected and processed for identification, serogrouping andtestingsusceptibilities for three antibiotics using standard microbiological techniques. Data onepidemiologic risk factors were collected using a structured questionnaire and the magnitude oftheir association with carriage was assessed using bivariate and multivariate analysis.Result: A total of 240 samples were collected (115 from males and 125 from females). Themean age of study participants was 11.1 years. The prevalence of nasal carriage for Neisseria meningitidis was 20.4% (49/240). Carriage was significantly higher among children living undercrowded conditions (OR1.268; 95% CI: 1.186 – 1.355; p = 0.006). The predominant serogroupswere W135 – 20/49 isolates (40.8%) and C - 12/49 isolates (24.5%) and 83.7% of meningococciwere sensitive for Ciprofloxacin. In contrast, isolates showed high resistance to Ceftriaxone(69.4%) while only 4.2% were sensitive for Penicillin. Multi-drug resistance was documented for14.3% of the isolates.Conclusions: Meningococcal carriage rate was found to be high with higher rates associatedwith children and adolescents living in crowded living conditions. Predominant isolates were ofserogroup W135 and C and the isolates showed marked susceptibility to Ciprofloxacin andresistance to Ceftriaxone and Penicillin.Keywords: N. meningitidis, Nasal carriage, Antibiotic susceptibility, Serogroup, Children,Adolescents, Ethiopia
Proper citation: Tinsae Alemayehu 2017. Nasal carriage rate and antibiotic susceptibility pattern of Neisseria meningitidis among healthy Ethiopian children and adolescents. protocols.io dx.doi.org/10.17504/protocols.io.jfbcjin Copy
Authors: Nara Gyzely de Morais Magalhães , Cristovam Guerreiro Diniz and Cristovam Wanderley Picanço Diniz
Summary: Protocol for Immunohistochemistry free-floating sections with anti-doublecortin antibody for avian tissue.
Proper citation: Nara Gyzely de Morais Magalhães , Cristovam Guerreiro Diniz and Cristovam Wanderley Picanço Diniz 2017. DCX Immunohistochemistry Protocol. protocols.io dx.doi.org/10.17504/protocols.io.h57b89n Copy
Authors: Robert Heuckeroth, Silvia Huerta Lopez, Kahleb Graham, Rajarshi Sengupta
Group: Optical Clearing of Tissue
Summary: INTRODUCTION: Bowel pathology is routinely evaluated by sectioning tissue, staining, and light microscopy. Tissue sectioning does not provide robust information about three-dimensional structures. Defining bowel anatomy in three dimensions is especially valuable for understanding human bowel motility disorders. Intrinsic mechanisms controlling bowel motility include the enteric nervous system (ENS), pacemaker cells called interstitial cells of Cajal, smooth muscle cells and PDGFRalpha+ cells. One problem is that the cells that control motility are distributed throughout the bowel wall and the bowel wall is too thick and opaque to permit direct visualization without sectioning.OBJECTIVES: To develop an efficient and reproducible method to make human colon translucent, stain with antibodies, and visualize cells that control bowel motility in three dimensions.METHODS: Tissue is cleaned, trimmed to remove fat, pinned flat, gently stretched and then fixed in 4% paraformaldehyde before storage. To begin clearing tissue is treated with 100% methanol, then permeabilized with Dent's bleach (Methanol, DMSO and hydrogen peroxide) and rinsed with PBS. Blocking is performed for 3 days at room temperature. Incubation with primary antibodies occurs for 14 days at 37 oC. Unbound primary antibody is washed out over the course of one day. Secondary antibody staining is performed for three days at 37 oC. After washing in PBS, tissue is dehydrated using a graded methanol series and then cleared using Murray's Clear (Benzyl Benzoate: Benzyl Alcohol). For confocal imaging, tissue is mounted in Murray's Clear.RESULTS: This approach provides efficient tissue clearing and reproducible staining with a subset of tested antibodies. We routinely obtain images using two or three antibodies and can visualize stained cells all the way through the bowel wall thickness. Confocal imaging permits excellent visualization and three dimensional reconstruction. We routinely stain 1 cm x 1 cm human colon pieces, but the same methods can be used to stain and image colon tissue many centimeters in length.CONCLUSION: We established an efficient and reproducible method for clearing, staining with antibodies and imaging colon tissue. This approach works well to generate three dimensional images of stained cells. We can visualize many cell types that control bowel motility, but the approach will also probably work to see other cell types with appropriate antibodies. Only a subset of antibodies work with this organic solvent based fixation and clearing method.
Proper citation: Robert Heuckeroth, Silvia Huerta Lopez, Kahleb Graham, Rajarshi Sengupta 2020. Human colon tissue clearing and Immunohistochemistry. protocols.io dx.doi.org/10.17504/protocols.io.wyeffte Copy
Authors: Karen Maciel de Oliveira
Summary: Superoxide dismutase (SOD) activity is evaluated by means of a previously described spectrophotometric method [48].
Proper citation: Karen Maciel de Oliveira 2018. Superoxide dismutase activity assessment. protocols.io dx.doi.org/10.17504/protocols.io.rund6ve Copy
Authors: Igem Dusseldorf
Proper citation: Igem Dusseldorf 2019. Western Blot (tank-blot) + Antibody staining. protocols.io dx.doi.org/10.17504/protocols.io.8gdhts6 Copy
Authors: Nicholas W. West
Proper citation: Nicholas W. West 2019. LAMP in situ complete. protocols.io dx.doi.org/10.17504/protocols.io.57cg9iw Copy
Authors: Vinay K Nagarajan, Monica Accerbi, Pamela J Green
Summary: This protocol was developed for transformation of Heterosigma akashiwo by Agrobacterium tumefaciens. H. akashiwo does not grow on solid medium, so transformants are grown "in bulk" and should not be considered clonal. We have had limited success with this protocol and may require further optimization.
Proper citation: Vinay K Nagarajan, Monica Accerbi, Pamela J Green 2017. Efforts to transform Heterosigma akashiwo using an Agrobacterium-mediated approach. protocols.io dx.doi.org/10.17504/protocols.io.jsxcnfn Copy
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