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Authors: Pradeep Rajendran, Guy Salama, Ching Zhu, Peter Hanna
Group: SPARC

Proper citation: Pradeep Rajendran, Guy Salama, Ching Zhu, Peter Hanna 2020. Dual Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart during Optogenetic Stimulation of the ICNS. protocols.io https://dx.doi.org/10.17504/protocols.io.bcdtis6n Copy   

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Authors: Shaina Robbins, Alison Moss, Sean Nieves
Group: SPARC
Summary: This protocol utilizes Fluidigm's Biomark system which performs high-throughput real-time PCR that can assay 48 or 96 genes for 48 or 96 samples respectfully. This protocol is used for gene expression targeting samples at the single-cell scale and can be used with the 48.48 Dynamic Array integrated fluidic circuit (IFC) or the 96.96 Dynamic Array IFC. Note that this protocol assumes that single-cell samples are captured using the Arcturus Laser Capture Microdissection system.

Proper citation: Shaina Robbins, Alison Moss, Sean Nieves 2020. BioMark Single Cell Protocol (Two-Step RTSTA). protocols.io https://dx.doi.org/10.17504/protocols.io.wdsfa6e Copy   

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Authors: Leah Reznikov
Group: SPARC
Summary: RNA Sequencing of Porine Ganglia and brainstem Animals. A total of 44 piglets (Yorkshire Landrace breed, 2–3 days of age) were fed commercial milk replacer (Soweena Litter Life) and allowed a 36–48-h acclimation period before interventions began. The University of Florida Animal Care and Use Committee approved all procedures. Procedures were completed in accordance with federal policies and guidelines. A total of 12 piglets were reserved for RNA sequencing experiments (6 male and 6 female). Airway instillation. After acclimation, piglets were anesthetized with 8% SevoThesia (Henry Schein). The piglets’ airways were accessed with a laryngoscope; a laryngotracheal atomizer (MADgic) was passed directly beyond the vocal folds, as previously described to aerosolize either a 500 μl 0.9% saline control or 1% acetic acid in 0.9% saline solution to the airway. This procedure results in widespread distribution of aerosolized solutions throughout the piglet airway, including the lung. Consistent with acid as a cough-evoking stimulus acetic acid induced cough in 90% or greater of the piglets. Nodose ganglia and brain stem isolation. Forty-eight hours after instillations, piglets were euthanized with a 90 mg/kg intravenous Euthasol solution (Henry Schein). Nodose ganglia were carefully dissected using methods that we previously developed. Ganglia were removed, placed in TRIzol, and stored at −80°C until RNA extraction. Any connective tissues, inflammatory cells, satellite cells, or blood cells covering the ganglia were not removed. A region of the brain stem encompassing the nucleus tractus solitarius, nucleus ambiguous, and dorsal motor nucleus was removed. Briefly, a blunt scissor was used to cut through the sagittal suture of the skull post mortem, and the brain was delicately excised. The cerebellum was removed, and the obex was identified. A section of the brain stem encompassing 2 mm rostral and 2 mm caudal to the obex was then removed and placed in RNAlater (Thermo Fisher Scientific). The block was further trimmed to extend 2 mm in depth and 2 mm lateral from the obex, while still submerged in RNAlater. RNA isolation. RNA from thenodose ganglia, and brain stem were isolated using RNeasy Lipid Tissue kit (Qiagen) with optional DNase digestion (Qiagen). RNA concentrations were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific). RNA sequencing. RNA concentration was determined on Qubit 2.0 Fluorometer (ThermoFisher/Invitrogen), RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies). Total RNA with 28S/18S > 1 and RNA integrity number (RIN) ≥ 7 were used for RNAseq library construction. RNAseq library were constructed using NEBNext Ultra Directional RNA library prep kit for Illumina (New England Biolabs) following the manufacturer’s recommendations. Briefly, 1,000 ng of total RNA was used for mRNA isolation using NEBNext Ploy(A) mRNA magnetic isolation module (New England Biolabs, cat. no. E7490). RNA library construction was achieved using NEBNext Ultra Directional RNA library prep kit for Illumina (New England Biolabs; cat. no. E7420). Thirty-six barcoded libraries were sized on the bioanalyzer and quantitated by QUBIT. Libraries were pooled in an equimolar ratio and sequenced by Illumina HiSeq 3000 2X100 cycles run for total of six runs (Illumina). RNA library construction was performed at the Interdisciplinary Center for Biotechnology Research (ICBR) Gene Expression & Genotyping Core, University of Florida (UF). HiSeq 3000 sequencing run was performed at the ICBR NextGen DNA Sequencing core, UF. The nodose ganglia and brain stem of three individual piglets for each condition were prepared separately and used. Mapping and differential gene expression. The quality of the RNA-Seq sequence data was first evaluated using FastQC before further downstream analysis. Low-quality sequences were removed, and the poor-quality part of the reads were trimmed using Trimmomatic. Star Aligner was used to map high-quality paired-end reads to Sus scrofa genome, Sscrofa11.1. Expression was obtained using RSEM. The expected read counts and fragments per kilobase of transcript per million-mapped reads (FPKM) were extracted for further analysis. The estimated read counts were taken as input for edgeR to perform differential expression analysis using generalized linear models. The threshold for calling significantly differentially expressed genes was set at FDR 0.05 with the average FPKM for at least one of each comparison group being higher than 0. Additional criteria using P This protocol accompanies the following publication 3. Reznikov LR, Liao YSJ, Gu T, Davis K, Kuan SP, Atanasova KR, Dadural JS, Collins EN, Guevara MV, Vogt KM (2019) Sex-specific airway hyperreactivity and sex-specific transcriptome remodeling in neonatal piglets challenged with intra-airway acid. AJP-Lung. Jan 1;316(1):L131-L143. doi: 10.1152/ajplung.00417.2018. Epub 2018 Nov 8. https://www.physiology.org/doi/full/10.1152/ajplung.00417.2018#

Proper citation: Leah Reznikov 2019. RNA Sequencing of Porine Ganglia and brainstem. protocols.io https://dx.doi.org/10.17504/protocols.io.w5sfg6e Copy   

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Authors: Shaina Robbins
Group: SPARC

Proper citation: Shaina Robbins 2020. Embedding Rat Heart. protocols.io https://dx.doi.org/10.17504/protocols.io.w52fg8e Copy   

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Authors: Shaina Robbins, Alison Moss
Group: SPARC
Summary: This is the standard procedure we use when sacrificing our Sprague-Dawley rats.

Proper citation: Shaina Robbins, Alison Moss 2020. Sacrificing Rat. protocols.io https://dx.doi.org/10.17504/protocols.io.ww6ffhe 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 2A 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 2A Receptor (5-HT2AR) Immunohistochemistry Protocol in Rat Tissues Labeled with Cholera Toxin B-fragment. protocols.io https://dx.doi.org/10.17504/protocols.io.2khgct6 Copy   

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Authors: Janet Keast, Peregrine Osborne, Nicole Wiedmann
Group: SPARC
Summary: This collection describes the procedures required to visualize and characterize lumbosacral spinal neurons that are activated by cystometry of awake adult male and female Sprague-Dawley rats. This collection includes protocols for:STAGE 1: Surgery to cannulate the bladder, followed by recovery then cystometrySTAGE 2: Intracardiac perfusion with fixative to preserve the spinal cord tissueSTAGE 3: Immunohistochemical labelling of spinal cord sections to visualise immediate early gene expression in specific spinal regions and neuronal populationsSTAGE 4: Microscopy and image analysis to assess patterns of immediate early gene expression in different spinal cord regions

Proper citation: Janet Keast, Peregrine Osborne, Nicole Wiedmann 2020. Immediate Early Gene (IEG) mapping of spinal cord neurons activated by cystometry-induced micturition in rats [keast-002]. protocols.io https://dx.doi.org/10.17504/protocols.io.bakxicxn Copy   

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Authors: J. Ashley Ezzell, Nicole A. Pelot, Kara A. Clissold, Warren M. Grill
Group: SPARC
Summary: Protocol for collection, histological processing, and imaging of rat vagus nerves.

Proper citation: J. Ashley Ezzell, Nicole A. Pelot, Kara A. Clissold, Warren M. Grill 2020. SPARC_Duke_PelotGrill_OT2-OD025340_RatVagusNerve_Collection_Histology_Microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.bh4bj8sn Copy   

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Authors: James Fallon, Sophie Payne, Janet Keast, Peregrine Osborne
Group: SPARC
Summary: A pelvic nerve array is custom designed for implantation onto the pelvic nerve of male rats. This device is used for long-term (recovery) experiments. The device can electrically stimulate the pelvic nerve as well as record electrically evoked or spontaneous neural activity. Previously, we have successfully implanted rats for 2 months with no reports of biological adverse complications or damage sustained to the array. The surgical procedure 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: James Fallon, Sophie Payne, Janet Keast, Peregrine Osborne 2020. Implantation of a pelvic nerve array in rats_original from SP. protocols.io https://dx.doi.org/10.17504/protocols.io.bfwxjpfn Copy   

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Authors: Janet Keast, Peregrine Osborne
Group: SPARC
Summary: This protocol describes immunohistochemical procedures applied to thick (50 µm) cryosections mounted directly on slides. It is used when the structures to be analysed are too large to remain intact within thin (10-20 µm) cryosections. Antibodies have been selected to distinguish different neurochemical classes of autonomic ganglion neurons and synaptic boutons associated with these neurons. The protocol can also be used to characterize neurons containing retrograde tracer.

Proper citation: Janet Keast, Peregrine Osborne 2020. Immunohistochemical labeling of thick cryosections from pelvic ganglia. protocols.io https://dx.doi.org/10.17504/protocols.io.bakcicsw Copy   

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Authors: James Fallon, Sophie Payne, Janet Keast, Peregrine Osborne
Group: SPARC
Summary: A pelvic nerve array is custom designed for implantation onto the pelvic nerve of male rats. This device is used for long-term (recovery) experiments. The device can electrically stimulate the pelvic nerve as well as record electrically evoked or spontaneous neural activity. Previously, we have successfully implanted rats for 2 months with no reports of biological adverse complications or damage sustained to the array. The surgical procedure 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: James Fallon, Sophie Payne, Janet Keast, Peregrine Osborne 2020. Implantation of a pelvic nerve array in rats. protocols.io https://dx.doi.org/10.17504/protocols.io.bjvxkn7n Copy   

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Authors: Nicole A. Pelot, Gabriel B. Goldhagen, Jake E. Cariello, Warren M. Grill
Group: SPARC
Summary: The protocol describes image segmentation and image analysis methods to quantify rat vagus nerve morphology from Masson's trichrome-stained nerve cross sections.

Proper citation: Nicole A. Pelot, Gabriel B. Goldhagen, Jake E. Cariello, Warren M. Grill 2020. SPARC_Duke_PelotGrill_OT2-OD025340_RatVagusNerve_Morphology. protocols.io https://dx.doi.org/10.17504/protocols.io.y6hfzb6 Copy   

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Authors: Sirisha Achanta
Group: SPARC
Summary: Single cell high-throughput qRT-PCR protocol combines high sensitivity technique of single cell qPCR with high-throughput qPCR technology that can generate data from 96 samples and 96 genes in a single experiment. It can be adapted for various sample types- cell culture, tissue samples and extracted RNA (10pg) and measured on traditional qPCR and high-throughput qPCR platforms. The workflow is comprised of four steps – cell lysis, reverse transcription, pre-amplification and qPCR. Key features of this protocol are; processing low input samples directly to reverse transcription without RNA extraction which minimizes sample loss, pre-amplification enables amplification of cDNA from single cells to detectable levels for qPCR and measuring up to 400 genes from a single cell sample/10pg of RNA (starting material). Robust reproducible and versatile this protocol can be adapted to several upstream and downstream techniques.

Proper citation: Sirisha Achanta 2020. SINGLE CELL HIGH-THROUGHPUT QRT-PCR PROTOCOL. protocols.io https://dx.doi.org/10.17504/protocols.io.bftdjni6 Copy   

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Authors: Shaina Robbins, Alison Moss, Sean Nieves
Group: SPARC
Summary: This collection of protocols were used to obtain the current data for the Blackfynn Dataset Molecular Phenotype Distribution of Single Rate ICN Neurons, which we refer to as Rat Heart B.

Proper citation: Shaina Robbins, Alison Moss, Sean Nieves 2020. Molecular Phenotype Distribution of Single Rat ICN Neurons - Heart B. protocols.io https://dx.doi.org/10.17504/protocols.io.w56fg9e Copy   

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Authors: J. Ashley Ezzell, Nicole A. Pelot, Kara A. Clissold, Warren M. Grill
Group: SPARC
Summary: Protocol for collection, histological processing (paraffin embedding, Masson's trichrome staining), and imaging of pig vagus nerves.

Proper citation: J. Ashley Ezzell, Nicole A. Pelot, Kara A. Clissold, Warren M. Grill 2020. SPARC_Duke_PelotGrill_OT2-OD025340_PigVagusNerve_Collection_Histology_Microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.6bqhamw Copy   

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Authors: Janet Keast, Peregrine Osborne, Nicole Wiedmann
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, Nicole Wiedmann 2020. Intracardiac perfusion with fixative for anatomical studies. protocols.io https://dx.doi.org/10.17504/protocols.io.bahzib76 Copy   

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Authors: Nicole A. Pelot, J. Ashley Ezzell, Gabriel B. Goldhagen, Kara A. Clissold, Warren M. Grill
Group: SPARC
Summary: The protocol describes immunohistochemistry with anti-claudin-1, imaging, image segmentation, and image analysis methods to quantify human vagus nerve morphology.

Proper citation: Nicole A. Pelot, J. Ashley Ezzell, Gabriel B. Goldhagen, Kara A. Clissold, Warren M. Grill 2020. SPARC_Duke_PelotGrill_OT2-OD025340_PigVagusNerve_FibronectinIF_Morphology. protocols.io https://dx.doi.org/10.17504/protocols.io.bfwtjpen Copy   

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Authors: Jeffrey Ardell
Group: SPARC
Summary: Neural analysis using linear micrarray electrodes.

Proper citation: Jeffrey Ardell 2019. Pig-Neural recording and analysis-workflow. protocols.io https://dx.doi.org/10.17504/protocols.io.2i4gcgw Copy   

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Authors: Elisa Gonzalez-Rothi, Latoya Allen, Alec Simon, Yasin Seven, Marissa Ciesla, Gordon Mitchell
Group: SPARC
Summary: This protocol describes the procedures for conducting long-term exposure to various intermittent hypoxia (or normoxia) protocols in rats.

Proper citation: Elisa Gonzalez-Rothi, Latoya Allen, Alec Simon, Yasin Seven, Marissa Ciesla, Gordon Mitchell 2019. SPARC Long-term exposure to intermittent hypoxia (or normoxia) using a custom in-cage computer controlled system. protocols.io https://dx.doi.org/10.17504/protocols.io.2j3gcqn Copy   

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Authors: Janet Keast, Peregrine Osborne, Nicole Wiedmann
Group: SPARC
Summary: This protocol is used for bladder cannulation and cystometry in an experimental adult male or female rat. The surgery 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, Nicole Wiedmann 2020. Cystometry in awake rats. protocols.io https://dx.doi.org/10.17504/protocols.io.bakjicun Copy   

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