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Authors: Elisa Gonzalez-Rothi, Marissa Ciesla, Latoya Allen, Gordon Mitchell
Group: SPARC
Summary: This protocol describes the procedure for performing a left lateral C2 spinal cord hemisection in rats

Proper citation: Elisa Gonzalez-Rothi, Marissa Ciesla, Latoya Allen, Gordon Mitchell 2019. SPARC C2 Spinal Cord Hemisection Protocol in Rats. protocols.io https://dx.doi.org/10.17504/protocols.io.2kngcve Copy   

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Authors: Martha Campbell Thompson, Malavika Nair
Group: Optical Clearing of Tissue, SPARC
Summary: Optical clearing of tissues to improve deep tissue microscopy was described over a century ago and methods have improved considerably following publication of the CLARITY method from Karl Deisseroth in 2013. Improvements in organic and inorganic methods are constantly reported for multiple species and organs. Large volume imaging was needed for human pancreas studies due to known heterogeneity in normal islet sizes and proportions of endocrine cells. Lobularity in islet beta-cell loss and islet inflammation is also seen in patients with type 1 diabetes. The iDISCO protocol allows for rapid clearing, easy staining, and high resolution confocal imaging. Human pancreas samples were cleared using iDISCO as originally published. This protocol describes steps to clear 4mm x 6mm x 400um deep sections of 4% paraformaldehyde fixed human pancreas using iDISCO-like reagents purchased from Visikol.

Proper citation: Martha Campbell Thompson, Malavika Nair 2020. Human Pancreas Optical Clearing by iDISCO and VISIKOL . protocols.io https://dx.doi.org/10.17504/protocols.io.ba4qigvw Copy   

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Authors: Janet Keast, Peregrine Osborne
Group: SPARC
Summary: This protocol is suitable for preserving tissues for anatomical studies of organs, ganglia, spinal cord or brain in adult rats. The protocol is performed under anesthesia and should incorporate all local requirements for standards of animal experimentation.

Proper citation: Janet Keast, Peregrine Osborne 2019. Intracardiac perfusion with fixative for anatomical studies [keast-001-stage02]. protocols.io https://dx.doi.org/10.17504/protocols.io.w3ffgjn Copy   

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Authors: Thomas Taylor-Clark, Seol-Hee Kim
Group: SPARC
Summary: Mice are euthanized, perfused with fixative and the vagal ganglia extracted. Vagal ganglia are then cyosectioned. Slices are stained for protein expression using immunohistochemistry. Expression of specific proteins and reporter proteins isarevisualized using microscopy.

Proper citation: Thomas Taylor-Clark, Seol-Hee Kim 2019. Dissection and immunohistochemistry of mouse vagal ganglia. protocols.io https://dx.doi.org/10.17504/protocols.io.w6cfhaw Copy   

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Authors: Shaina Robbins, Sean Nieves
Group: SPARC
Summary: This is the straightforward procedure we follow when sectioning rat heart.

Proper citation: Shaina Robbins, Sean Nieves 2020. Sectioning Rat Heart. protocols.io https://dx.doi.org/10.17504/protocols.io.w53fg8n Copy   

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Authors: Janet Keast, Peregrine Osborne
Group: SPARC
Summary: This collection describes the procedures required to visualise and characterize synaptic boutons associated with functionally classified pelvic ganglion autonomic neurons of adult male and female Sprague-Dawley rats. This collection includes protocols for:STAGE 1: Surgery to micro-inject fluorescent retrograde tracer dyes into one or more sites within the lower urinary tractSTAGE 2: Intracardiac perfusion with fixative to preserve neural tissues of interestSTAGE 3: Immunohistochemical labeling of thick cryosections of pelvic gangliaSTAGE 4: Confocal microscopy and image analysis of synaptic boutons associated with ganglion neurons.

Proper citation: Janet Keast, Peregrine Osborne 2020. Light microscopic analysis of synaptic input to neurons in the rat major pelvic ganglion [keast-003]. protocols.io https://dx.doi.org/10.17504/protocols.io.bakiicue Copy   

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Authors: Colleen clancy, Pei-Chi, Parya Aghasafari
Group: SPARC
Summary: Multi-scale computational modeling is a major branch of computational biology as evidenced by the US federal interagency Multi-Scale Modeling Consortium and major international projects. It invariably involves specific and detailed sequences of data analysis and simulation, often with multiple tools and datasets, and the community recognizes improved modularity, reuse, reproducibility, portability and scalability as critical unmet needs in this area. Scientific workflows are a well-recognized strategy for addressing these needs in scientific computing. While there are good examples if the use of scientific workflows in bioinformatics, medical informatics, biomedical imaging and data analysis, there are fewer examples in multi-scale computational modeling in general and cardiac electrophysiology in particular. Cardiac electrophysiology simulation is a mature area of multi-scale computational biology that serves as an excellent use case for developing and testing new scientific workflows. In this dataset, we develop, describe and test a computational workflow that serves as a proof of concept of a platform for the robust integration and implementation of a reusable and reproducible multi-scale cardiac cell and tissue model that is expandable, modular and portable. The workflow described leverages Python and Kepler-Python actor for plotting and pre/post-processing. During all stages of the workflow design, we rely on freely available open-source tools, to make our workflow freely usable by scientists.

Proper citation: Colleen clancy, Pei-Chi, Parya Aghasafari 2019. A multi-scale model of cardiac electrophysiology. protocols.io https://dx.doi.org/10.17504/protocols.io.5nkg5cw Copy   

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Authors: Lixin Wang, Pu-Qing Yuan, Honghui Liang, Yvette Tache
Group: SPARC
Summary: A table listed the information of antibodies used in the mouse colon. The protocol described the methods of colon tissues sampling, whole mount preparations, passive clarity technique, and respective fluorescent Immunohistochemistry for the antibody tests in different tissue preparations.

Proper citation: Lixin Wang, Pu-Qing Yuan, Honghui Liang, Yvette Tache 2021. Immunofluorescent methods for antibody test in mouse colon. protocols.io https://dx.doi.org/10.17504/protocols.io.bqi2muge Copy   

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Authors: Martha Campbell Thompson
Group: Human Cell Atlas Method Development Community, Optical Clearing of Tissue, SPARC
Summary: DRAFTPurpose:The purpose of this protocol is to outline proper procedures for fixed specimen processing using an automatic paraffin processor and sectioning and staining for histopathological review of normalcy. Scope:This protocol applies to human pancreas specimens that will be processed to paraffin blocks for HuBMAP Tissue Mapping Centers.Expected Outcome:Pancreas samples are processed properly and efficiently to whole slide images for histopathology review. Nuclei should stain blue. Cytoplasm of acinar cells should stain pink with blue zymogen granules. The large blood vessels should demonstrate clear differentiation of RBCs when present, surrounding collagen, and endothelial nuclei with scant cytoplasm.

Proper citation: Martha Campbell Thompson 2021. Human Pancreas Histopathology Assessment. protocols.io https://dx.doi.org/10.17504/protocols.io.br7tm9nn Copy   

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Authors: Elisa Gonzalez-Rothi, Arash Tadjalli, Raphael Perim, Gordon Mitchell
Group: SPARC
Summary: Protocol for conducting bilateral phrenic neurograms in anesthetized, paralyzed, vagotomized and mechanically ventilated rats - moderate acute intermittent hypoxia-induced phrenic long-term facilitation protocol

Proper citation: Elisa Gonzalez-Rothi, Arash Tadjalli, Raphael Perim, Gordon Mitchell 2019. SPARC bilateral terminal phrenic neurophysiology preparation with moderate acute intermittent hypoxia. protocols.io https://dx.doi.org/10.17504/protocols.io.2jpgcmn Copy   

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Authors: Maci Heal
Group: SPARC

Proper citation: Maci Heal 2021. Neurolucida 360: Detecting muscle fiber orientation . protocols.io https:// Copy   

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Authors: Elisa Gonzalez-Rothi, Yasin Seven, Latoya Allen, Marissa Ciesla, Gordon Mitchell
Group: SPARC
Summary: This protocol describes the immunofluorescent labeling technique used to identify serotonin 2B receptor expression in CtB-labelled phrenic motor neurons and within a defined region of interest surrounding phrenic motor neurons.

Proper citation: Elisa Gonzalez-Rothi, Yasin Seven, Latoya Allen, Marissa Ciesla, Gordon Mitchell 2019. SPARC Serotonin 2B Receptor (5-HT2BR) Immunohistochemistry Protocol in Rat Tissues Labeled with Cholera Toxin B-fragment. protocols.io https://dx.doi.org/10.17504/protocols.io.2kigcue Copy   

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Authors: Clara Leung, Maci Heal, Shaina Robbins, Alison Moss, Corey Monteith, Susan Tappan
Group: SPARC
Summary: This protocol describes the process of using the Tissue Mapper software to map single neurons of the intrinsic cardiac nervous system, annotate or "trace" key cardiac anatomy on select histological sections of an image volume, and to visualize the mapped neurons and traced anatomy in a 3D reconstructed heart.

Proper citation: Clara Leung, Maci Heal, Shaina Robbins, Alison Moss, Corey Monteith, Susan Tappan 2020. Single-Cell ICN Neuron Mapping and 3D Heart Reconstruction with Tissue Mapper. protocols.io https://dx.doi.org/10.17504/protocols.io.bdz5i786 Copy   

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Authors: Christina M Wright, Robert O Heuckeroth
Group: SPARC
Summary: INTRODUCTION: The human enteric nervous system (ENS) is a complex network of neurons and glia that extends throughout the length of the bowel. There are many neuron and glia types, but very little is known about the human ENS compared to other species. There are two main ENS layers. The myenteric plexus neurons are clustered into ganglia with thick nerve fiber bundles running between ganglia. Submucosal neuron ganglia are scattered throughout the region between circular muscle and the epithelial lining of the bowel. One key problem with studying human ENS is that these cells are a very minor component of the bowel wall (OBJECTIVES: To obtain a suspension of single nuclei from human colon myenteric plexus that could be used for RNA-seq.METHODS: 1) Human colon "myenteric plexus" is dissected from the bowel wall after labeling with a live cell dye and then frozen in OCT. 2) Frozen "myenteric plexus" is sectioned on a cryostat to generate small fragments and disrupt muscle and connective tissue. 3) RNA quality is assessed from a few sections using an Agilent Bioanalyzer. 4) Nuclei are obtained from frozen sections using Dounce homogenization and labeled with Hoechst 33342 dye. 5). Nuclei are separated from other cell debris by FACS. 6) RNA-seq data were obtained using the 10X Genomic single cell sequencing platform.RESULTS: Live dye labeling works well on living tissue to visualize the ENS in human colon. Careful dissection is required to micro-dissect "myenteric plexus" from from within bowel wall muscle layers. A suspension of single nuclei is efficiently isolated by freezing isolated tissue in OCT, frozen sectioning, Dounce homogenizing, and FACS sorting Hoechst 33342 stained nuclei. The isolated nuclei are suitable for sequencing on the 10X Genomics single cell sequencing platform. This procedure dramatically enriched for cells in the region of the myenteric plexus, but unfortunately only a small percentage of the cells isolated are myenteric neurons. In addition to myenteric neurons, we obtained data from enteric glia, smooth muscle, muscularis macrophage, interstitial cells of Cajal, and PDGFRalpha+ cells that are known to be closely associated with myenteric plexus.CONCLUSION: We tried many approaches to optimize this procedure including additional steps to enrich for myenteric neuron nuclei. These additional procedures invariably reduced total cell yield without enriching significantly for neuronal nuclei. We do not know yet why neuronal nuclei remain a small component of our total nuclei population, but suspect that neuronal nuclei are more fragile than other nuclei isolated. This approach generated valuable data at a single nucleus RNA-seq level for all cell types within the muscle wall known to control bowel motility.

Proper citation: Christina M Wright, Robert O Heuckeroth 2021. Processing Human Colon "Myenteric Plexus" for Single Nuclei RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.w26fghe Copy   

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Authors: Pu-Qing Yuan, Yvette Taché
Group: SPARC

Proper citation: Pu-Qing Yuan, Yvette Taché 2019. Tache_Yuan_OT2OD024899_CLARITYAnd3DImagingOfColonicENSintheMouseAndPig_1_2019-Pig_Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.4r9gv96 Copy   

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Authors: Pu-Qing Yuan, Lixin Wang, Yvette Taché
Group: SPARC

Proper citation: Pu-Qing Yuan, Lixin Wang, Yvette Taché 2019. Tache_Yuan_OT2OD024899_CLARITYAnd3DImagingOfColonicENSintheMouseAndPig_1_2019-Mouse_Protocol (Annotation Copy). protocols.io https://dx.doi.org/10.17504/protocols.io.4sagwae Copy   

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Authors: Christina M Wright, Robert O Heuckeroth
Group: SPARC
Summary: ABSTRACTINTRODUCTION: The human enteric nervous system (ENS) is a complex network of neurons and glia that extends throughout the length of the bowel. There are many neuron and glia types, but very little is known about the human ENS compared to other species. There are two main ENS layers. The myenteric plexus neurons are clustered into ganglia with thick nerve fiber bundles running between ganglia. Submucosal neuron ganglia are scattered throughout the region between circular muscle and the epithelial lining of the bowel. One key problem with studying human ENS is that these cells are a very minor component of the bowel wall (< 1: 10,000 cells) and they are surrounded by other tissue. The bowel wall is not easily dissociated making it challenging to isolate cells of the human ENS for any type of analsysis. OBJECTIVES: To enrich for neurons from human colon myenteric plexus using PHOX2B antibody and FACS METHODS: 1) Human colon muscle was dissected from the bowel wall and then frozen in OCT. 2) Frozen muscle was sectioned on a cryostat to generate small fragments and disrupt muscle and connective tissue. 3) RNA quality was assessed from a few sections using an Agilent Bioanalyzer. 4) Nuclei were obtained from frozen sections using Dounce homogenization and stained with antibody to PHOX2B. 5). Nuclei were separated from other cell debris by FACS. 6) RNA-seq data were obtained using the 10X Genomic single cell sequencing platform. RESULTS: This procedure worked well to isolate nuclei from human colon muscle. PHOX2B antibody labeling led to a small population of nuclei that appeared to be antibody stained under the microscope and have attached antibody by FACS. Unfortunately, single nucleus RNA-seq demonstrated that we had not enriched for myenteric neurons with this approach. RNA-seq data were obtained from many other cell types within the muscle layers of the bowel wall. CONCLUSION: This approach generated valuable data at a single nucleus RNA-seq level for many cell types within the muscle wall known to control bowel motility, but did not enrich for neuronal nuclei, even though PHOX2B antibody selectively stains myenteric plexus nuclei within the human bowel myenteric plexus

Proper citation: Christina M Wright, Robert O Heuckeroth 2021. Processing Human Colon muscle layers for Single Nuclei RNA-seq using PHOX2B antibody immunoselection. protocols.io https://dx.doi.org/10.17504/protocols.io.w3afgie Copy   

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Authors: Zachariah Sperry, Kyounghwan Na, Mihaly Vöröslakos, Saman Parizi, James Jun, Tim M. Bruns, Euisik Yoon, John P. Seymour
Group: SPARC
Summary: Dorsal root ganglia (DRG) are components of spinal roots containing sensory cell bodies, and hold significant promise as neural recording sites for sensory neuroprostheses.This protocol details the fabrication and use of a high-density flexible polyimide electrode array into the sacral DRG of an anesthetized feline for neural recordings.

Proper citation: Zachariah Sperry, Kyounghwan Na, Mihaly Vöröslakos, Saman Parizi, James Jun, Tim M. Bruns, Euisik Yoon, John P. Seymour 2021. High-Density Penetrating Microelectrode Recordings from Anesthetized Feline Dorsal Root Ganglia. protocols.io https://dx.doi.org/10.17504/protocols.io.w5nfg5e Copy   

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Authors: Linda J. Fothergill, Martin Stebbing, Billie Hunne, Giorgia Galiazzo, Josiane Fahkry, Frank Weissenborn, Therese Fazio Coles, John B. Furness
Group: SPARC

Proper citation: Linda J. Fothergill, Martin Stebbing, Billie Hunne, Giorgia Galiazzo, Josiane Fahkry, Frank Weissenborn, Therese Fazio Coles, John B. Furness 2021. Immunohistochemistry and high resolution microscopy of pig gastric enteroendocrine cells. protocols.io https:// Copy   

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Authors: Janet Keast, Peregrine Osborne
Group: SPARC
Summary: This protocol is used to visualise sensory and autonomic neurons innervating the bladder body (dome), bladder trigone or proximal urethra in an experimental adult male or female rat. The protocol is performed under anesthesia and should incorporate all local requirements for standards of animal experimentation, including methods of anesthesia, surgical environment, and post-operative monitoring and care.

Proper citation: Janet Keast, Peregrine Osborne 2019. Use of tracer dyes to label neural projections to lower urinary tract organs [keast-001-stage01]. protocols.io https://dx.doi.org/10.17504/protocols.io.w3dfgi6 Copy   

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