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Authors: Paola Marcovecchio, Sara Mcardle, Zbigniew Mikulski, Angela Denn, Katarzyna Dobaczewska
Group: Optical Clearing of Tissue, La Jolla Institute Microscopy Core
Summary: Fixation of mouse tissues for either histology or fluorescence microscopy is an essential first step in tissue preparation. In this protocol, we outline a reliable method for fixation and perfusion of mice for tissue histology or microscopy. Tissues prepared by this method can be used downstream as FFPE sections, cryosections for fluorescence microscopy, or tissue clearing for whole tissue imaging. For mice with transgenic fluorescent reporters, it is especially important that the tissues are preserved properly to ensure that the signal of the fluorescent reporter can be imaged.

Proper citation: Paola Marcovecchio, Sara Mcardle, Zbigniew Mikulski, Angela Denn, Katarzyna Dobaczewska 2018. Mouse Tissue Fixation with Paraformaldehyde for Fluorescent Reporter Mice. protocols.io https://dx.doi.org/10.17504/protocols.io.vnge5bw Copy   

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Authors: Joao Vitor Molino
Summary: This protocols describe the steps required for the preparation of agarose pads for live cell fluorescent microscopy.

Proper citation: Joao Vitor Molino 2020. Agarose pads for microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.bkn8kvhw Copy   

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Authors: Jiri Hulcr, Andrew J. Johnson, Demian F Gomez
Group: Protocols Bark Beetle Mycobiome
Summary: This protocol describes the different methods to collect and preserve bark ambrosia beetles. This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 https://doi.org/10.1007/s13199-020-00686-9.

Proper citation: Jiri Hulcr, Andrew J. Johnson, Demian F Gomez 2020. Collecting Bark and Ambrosia Beetles. protocols.io https://dx.doi.org/10.17504/protocols.io.bpjdmki6 Copy   

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Authors: Anna Lyubimova, Shalev Itzkovitz, Jan Philipp Junker, Zi Peng Fan, Xuebing Wu, Alexander van Oudenaarden
Group: Human Cell Atlas Method Development Community
Summary: We present a protocol for visualizing and quantifying single mRNA molecules in mammalian (mouse and human) tissues. In the approach described here, sets of about 50 short oligonucleotides, each labeled with a single fluorophore, are hybridized to target mRNAs in tissue sections. Each set binds to a single mRNA molecule and can be detected by fluorescence microscopy as a diffraction-limited spot. Tissue architecture is then assessed by counterstaining the sections with DNA dye (DAPI), and cell borders can be visualized with a dye-coupled antibody. Spots are detected automatically with custom-made software, which we make freely available. The mRNA molecules thus detected are assigned to single cells within a tissue semiautomatically by using a graphical user interface developed in our laboratory. In this protocol, we describe an example of quantitative analysis of mRNA levels and localization in mouse small intestine. The procedure (from tissue dissection to obtaining data sets) takes 3 d. Data analysis will require an additional 3–7 d, depending on the type of analysis.

Proper citation: Anna Lyubimova, Shalev Itzkovitz, Jan Philipp Junker, Zi Peng Fan, Xuebing Wu, Alexander van Oudenaarden 2018. Single-molecule mRNA detection and counting in mammalian tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.mc7c2zn Copy   

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Authors: Mohammad Farris Iman Leong Bin Abdullah, Kok Leng Tan, Salbiah Mohd Isa, Nur Sabrina Yusoff, Nelson Jeng Yeou Chear, Darshan Singh
Summary: The protocol describe the steps involved in conducting fasting lipid profile and liver function test from the blood samples of regular kratom users and healthy non-kratom using subjects in this study. This tests were conducted in an ISO 15189-certified laboratory at Advanced Medical and Dental Institute, Universiti Sains Malaysia. The blood parameters which were determined quantitatively included serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL), total protein (TP), albumin, alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST), total bilirubin (TB) and direct bilirubin (DB). While globulin was determined from simple subtraction of albumin from total protein value, indirect bilirubin was determined by subtraction of direct bilirubin from total bilirubin value, and low-density lipoprotein (LDL) was calculated via Friedwald equation.

Proper citation: Mohammad Farris Iman Leong Bin Abdullah, Kok Leng Tan, Salbiah Mohd Isa, Nur Sabrina Yusoff, Nelson Jeng Yeou Chear, Darshan Singh 2020. Lipid profile of regular kratom (Mitragyna speciosa Korth.) users in the community setting. protocols.io https://dx.doi.org/10.17504/protocols.io.bfuwjnxe Copy   

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Authors: Yang Sun, David Lee, Jimmie Ye
Group: Human Cell Atlas Method Development Community
Summary: Purpose: To purify PBMCs from 20 or 50mL whole blood (per donor). SepMate tubes will be used with Lymphoprep to ease processing time and effort.

Proper citation: Yang Sun, David Lee, Jimmie Ye 2020. Whole Blood Processing (SepMate). protocols.io https://dx.doi.org/10.17504/protocols.io.ba87ihzn Copy   

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Authors: Dusan Velickovic, Jessica Lukowski, Guanshi Zhang, Theodore Alexandrov, Chris Anderton, Kumar Sharma
Group: KPMP
Summary: Mass spectrometry imaging is an exciting technologywhich enables a simultaneous analysis of multiple molecular components directly from single cells, tissues, and organs,. In combination with histological methods, this technique provides information about the spatial distribution of molecules in various biological tissues. Particularly, MALDI-MS imaging increases the coverage of metabolites by using different matrices and ion modes. In coordination with in situ analysis of proteins, transcripts and epigenetic marks, the complementary spatial information on metabolites will establish metabolic pathways that are dominant and characteristic of disease states. We have recently developed and optimized a spatial metabolomics approach to image small molecules in human kidneys and biopsy sized material. With our combined expertise at UTHSA, PNNL and EMBL and recent advances, we have established methods for identifying metabolites in human kidneys, employed ultra-high mass resolution MS imaging for tissue analysis, and developed a bioinformatics resource (METASPACE) to annotate metabolites for anatomical localization and 3-D reconstruction. Our integrated technology can easily connect with other TIS sites to provide biochemical readouts of genes/proteins in specific tissue and cellular compartments.Although METASPACE bioinformatics provides metabolite annotations of given reliability, there may be an inherent level of ambiguity due to the lack of untargeted MS/MS fragmentation in MALDI-MSI. Metabolite annotations from MALDI-MSI and METASPACE are structurally validated using orthogonal analytical techniques such as LC-MS/MS due to its high metabolome coverage, sensitivity, and throughput. The work flow for ‘bulk omics’ is shown in Figure 5.

Proper citation: Dusan Velickovic, Jessica Lukowski, Guanshi Zhang, Theodore Alexandrov, Chris Anderton, Kumar Sharma 2020. Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS) for Bulk Metabolomics. protocols.io https://dx.doi.org/10.17504/protocols.io.be2ajgae Copy   

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Authors: Mario Thomas, Jasmine Sollen, Natalia Ivanova, Michelle Feng, Reda Fayek, Heidi Abdilla, Stephanie Lim, Amanda Naaum, Holly Rumery, Mario Escobar
Group: Coronavirus Method Development Community, SANATA, InnOvOtion By Holly, InnOvOtion by Holly
Summary: This procedure outlines the protocol for testing for SARS-CoV-2 using a saliva sample collected from an individual. The purpose of this test is to detect low levels of SARS-CoV-2 antigen at a higher sensitivity. Precision Biomonitoring Inc. developed an ultra-rapid digital, disposable, highly-sensitive and inexpensive testing device used for screening purposes. The mobile app complementary to this medical device is connected through Bluetooth. Using this innovation, the user can be tested at point-of-care (POC) by a health care professional, and obtain qualitative results.

Proper citation: Mario Thomas, Jasmine Sollen, Natalia Ivanova, Michelle Feng, Reda Fayek, Heidi Abdilla, Stephanie Lim, Amanda Naaum, Holly Rumery, Mario Escobar 2021. XPRIZE SANATA Protocol for Saliva LFIA Test. protocols.io https://dx.doi.org/10.17504/protocols.io.brgsm3we Copy   

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Authors: Cancer Research UK / Wellcome Gurdon Institute media kitchen
Summary: Prepare 2L of B-Broth for culturing bacteria

Proper citation: Cancer Research UK / Wellcome Gurdon Institute media kitchen 2019. Prepare 2L of B-Broth for culturing bacteria. protocols.io https://dx.doi.org/10.17504/protocols.io.5spg6dn Copy   

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Authors: Ka-young Ban, D Osborn, Y Hameed, S Pandey, J Perez, PB Jones, JB Kirkbride
Summary: Aim: Personality Disorders (PD) often share clinical and phenomenological overlap with psychotic disorders, especially at onset. However, there is little research on comorbid PD among people experiencing first episode psychosis. We examined the prevalence of PD recording and its sociodemographic and clinical correlates in people accepted to Early Intervention in Psychosis (EIP) services. Methods: Participants were aged 16-35, accepted into 6 EIP services for suspected psychosis, as part of the Social Epidemiology of Psychoses in East Anglia (SEPEA) study. PD was recorded by clinicians according to ICD-10. Multilevel logistic regression was performed. Results: Of 798 participants, 76 people (9.5%) received a clinical diagnosis of PD, with emotionally unstable PD (75.0%, N=57) the most common subtype. In multivariable analysis, risk factors for PD included female sex (odds ratio [OR]: 3.4; 95% CI: 2.0-5.7), absence of psychotic disorder after acceptance to EIP (OR: 3.0; 95% CI: 1.6-5.5), more severe hallucinations (OR: 1.6; 95% CI: 1.2-2.1), and lower parental SES (OR: 1.4; 95% CI: 1.1-1.8). Compared with the white British, black and minority ethnic groups were less likely to receive a PD diagnosis (OR: 0.3; 95% CI: 0.1-0.7). There was no association between PD and neighbourhood-level deprivation or population-density. Conclusions: Recording of a PD diagnosis was three times more common amongst participants later found not to meet threshold criteria for psychotic disorder, implying phenomenological overlap at referral which highlights difficulties encountered in accurate diagnostic assessment, treatment and onward referral. People with PD experienced more individual-level, but not neighbourhood-level social disadvantage in an already disadvantaged sample.

Proper citation: Ka-young Ban, D Osborn, Y Hameed, S Pandey, J Perez, PB Jones, JB Kirkbride 2020. Personality disorder in an Early Intervention Psychosis cohort: Findings from the Social Epidemiology of Psychoses in East Anglia (SEPEA) study. protocols.io https://dx.doi.org/10.17504/protocols.io.bf3hjqj6 Copy   

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Authors: Jiao Wang

Proper citation: Jiao Wang 2017. Cell culture of 7721 or HeLa cells. protocols.io https://dx.doi.org/10.17504/protocols.io.iaqcadw Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the procedure for removal and embedding of the post-natal mouse brain.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. Brain Dissection of Post-natal Mice. protocols.io https://dx.doi.org/10.17504/protocols.io.besnjede Copy   

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Authors: Angel Justiz-Vaillant, Monica F. Smikle
Group: University of the West Indies, [email protected]
Summary: Peptostreptococcal protein L is an immunoglobulin-binding protein that interacts with the Fab region of many mammalian immunoglobulins [1].References1.Vaillant AJ, McFarlane-Andersonv N, Wisdom B, Mohammed W, Vuma S, et al. (2013) Immunoglobulin-binding Bacterial Proteins (IBP) Conjugates and their Reactivity with Immunoglobulin in Enzyme-Linked Immunosorbent Assays (ELISA). J Anal Bioanal Tech 4: 175. doi:10.4172/2155-9872.1000175

Proper citation: Angel Justiz-Vaillant, Monica F. Smikle 2020. Direct ELISA for investigating the binding of Peptostreptococcal Protein-L to immunoglobulins.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjxtkpnn Copy   

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Authors: Sierra Simpson, Olivier George
Group: George Lab, Metabolomics Protocols & Workflows

Proper citation: Sierra Simpson, Olivier George 2020. GNPS Untargeted Metabolomics Workflow . protocols.io https:// Copy   

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Authors: Kaichang Cheng, Xuemin Cheng, Yuqi Wang, Hongsheng Bi, Mark C. Benfield
Summary: This is an automatic plankton image recognition and enumeration system using an enhanced Convolutional Neural Network (CNN) and examined the performance of different network structures on automatic plankton image classification.

Proper citation: Kaichang Cheng, Xuemin Cheng, Yuqi Wang, Hongsheng Bi, Mark C. Benfield 2019. Enhanced convolutional neural network for plankton identification and enumeration. protocols.io https://dx.doi.org/10.17504/protocols.io.2u5gey6 Copy   

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Authors: Helen Smith, portelaa , Chloe Harvey
Group: WHO discharge readiness
Summary: Despite existence of global recommendations on postnatal care of mothers and newborns, care provided following childbirth is limited and often fails to address mothers’ concerns about herself and her baby. Women report leaving facilities without sufficient knowledge or skills to take care of themselves and their newborns. Women experience physical and emotional changes during the postnatal period and understanding women’s needs at this time is important for her subsequent health and the health of the newborn. Successful discharge happens when service providers meet the identified postnatal needs of women, caregivers and newborns.The World Health Organization is currently developing guidance on postnatal care. One aspect of the guidance relates to readiness for women and their newborns to be discharged from a facility after birth and the steps the health providers should take to prepare a woman, her newborn and parents/caregivers for discharge. However not much is known about the current state of discharge preparation and readiness practices in order to make recommendations for global implementation.As yet there is no comprehensive map of the evidence base; very little is known about working definitions and conceptual boundaries, what criteria for discharge readiness are in use or what strategies or interventions exist for improving discharge preparation and readiness.This scoping review will identify and map the range of policies, guidelines and literature on discharge readiness, clarify definitions of the concepts of discharge preparedness and discharge readiness, list criteria and items currently used to assess discharge readiness, and report on any tools, processes, strategies or other interventions that have been developed and/or implemented to improve discharge preparation and readiness.

Proper citation: Helen Smith, portelaa , Chloe Harvey 2020. Discharge preparation and discharge readiness in facilities prior to discharge after birth: a scoping review of the global policies, guidelines and literature. protocols.io https://dx.doi.org/10.17504/protocols.io.bpzymp7w Copy   

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Authors: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino
Summary: Separation and purification of PBMC from BUFFY COAT: list of published work using this protocolKustrimovic, N., Comi, C., Magistrelli, L., Rasini, E., Legnaro, M., Bombelli, R., Aleksic, I., Blandini, F., Minafra, B., Riboldazzi, G., Sturchio, A., Mauri, M., Bono, G., Marino, F., & Cosentino, M. (2018). Parkinson's disease patients have a complex phenotypic and functional Th1 bias: cross-sectional studies of CD4+ Th1/Th2/T17 and Treg in drug-naïve and drug-treated patients. Journal of neuroinflammation, 15(1), 205. https://doi.org/10.1186/s12974-018-1248-8Kustrimovic, N., Rasini, E., Legnaro, M., Bombelli, R., Aleksic, I., Blandini, F., Comi, C., Mauri, M., Minafra, B., Riboldazzi, G., Sanchez-Guajardo, V., Marino, F., & Cosentino, M. (2016). Dopaminergic Receptors on CD4+ T Naive and Memory Lymphocytes Correlate with Motor Impairment in Patients with Parkinson's Disease. Scientific reports, 6, 33738. https://doi.org/10.1038/srep33738Cosentino M., Ferrari M., Kustrimovic N., Rasini E., Marino F. (2015). Influence of dopamine receptor gene polymorphisms on circulating T lymphocytes: A pilot study in healthy subjects. Human immunology, 76, 10, 747-752. https://doi.org/10.1016/j.humimm.2015.09.032Kustrimovic, N., Rasini, E., Legnaro, M., Bombelli, R., Aleksic, I., Blandini, F., Comi, C., Mauri, M., Minafra, B., Riboldazzi, G., Sanchez-Guajardo, V., Marino, F., & Cosentino, M. (2016). Dopaminergic Receptors on CD4+ T Naive and Memory Lymphocytes Correlate with Motor Impairment in Patients with Parkinson's Disease. Scientific reports, 6, 33738. https://doi.org/10.1038/srep33738Cosentino M., Ferrari M., Kustrimovic N., Rasini E., Marino F. (2015). Influence of dopamine receptor gene polymorphisms on circulating T lymphocytes: A pilot study in healthy subjects. Human immunology, 76, 10, 747-752. https://doi.org/10.1016/j.humimm.2015.09.032

Proper citation: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. Separation and purification of human PBMC from BUFFY COAT. protocols.io https://dx.doi.org/10.17504/protocols.io.bc5kiy4w Copy   

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Authors: Nikki Freed, Olin Silander
Group: Coronavirus Method Development Community
Summary: To enable faster, easier sequencing of SARS-COV2 genomes with fewer steps than current methods, we use multiplexed 1200 base pair PCR amplicons with the Oxford Nanopore RAPID barcoding kit (RBK004). This is a modification of the ARTIC amplicon V3 sequencing protocol for MinION for nCoV-2019 developed by Josh Quick, which produces 400 base pair amplicons and uses the Oxford Nanopore Ligation barcoding kit (LSK-109). We have increased the size of the amplicons to 1200bp and use the RAPID barcode kit (RBK004), which enables requires less time and fewer reagents than the LSK-109 protocol. The amplicons produced in this protocol could also be used for Illumina sequencing. Primers were all designed using Primal Scheme: http://primal.zibraproject.org/, described here https://www.nature.com/articles/nprot.2017.066.Primer sequences are here: https://docs.google.com/spreadsheets/d/1M5I_C56ZC8_2Ycgm9EFieVlVNqxsP7dXAnGoBZy3nDo/edit?usp=sharingWe can ship a small amount of pooled primers to interested labs for further testing, email [email protected] or [email protected]

Proper citation: Nikki Freed, Olin Silander 2020. nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon). protocols.io https://dx.doi.org/10.17504/protocols.io.bgc8jszw Copy   

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Authors: Maira Rivera, Javiera Reyes, Paula Blazquez-Sanchez, Cesar A Ramirez-Sarmiento
Group: Reclone.org (The Reagent Collaboration Network)
Summary: This protocol has been optimized for the recombinant expression of codon-optizimed M-MLV and MashUp RT.The goal of this protocol was to eliminate the use of large volumes for dyalisis and potential issues with the protein crashing out of the solution due to the use of concentrators for buffer exchange of this enzyme into storage conditions. M-MLV RT has been further functionally tested and works as expected. The sequence of the plasmid encoding the codon-optimized M-MLV RT enzyme can be found here.

Proper citation: Maira Rivera, Javiera Reyes, Paula Blazquez-Sanchez, Cesar A Ramirez-Sarmiento 2021. Recombinant expression and purification of codon-optimized M-MLV and Mashup. protocols.io https://dx.doi.org/10.17504/protocols.io.bsernbd6 Copy   

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Authors: Bryce Killingsworth, Joshua Welsh, Tim Traynor, Michelle Pleet, Jennifer Jones
Group: Translational Nanobiology Section
Summary: This is a generic protocol for using the Spectradyne nCS1 resestive pulse sensing instrument to measure the diameter distribution and concentration of nanoparticles.

Proper citation: Bryce Killingsworth, Joshua Welsh, Tim Traynor, Michelle Pleet, Jennifer Jones 2020. Spectradyne nCS1: Sample measurement and device maintenence protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bmx8k7rw Copy   

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