Mammalia

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    Protocols in Current Issue
    Isolation of Nuclei from Mouse Dorsal Root Ganglia for Single-nucleus Genomics
    Authors:  Lite Yang, Ivan Tochitsky, Clifford J. Woolf and William Renthal, date: 08/05/2021, view: 24, Q&A: 0

    Primary somatosensory neurons, whose cell bodies reside in the dorsal root ganglion (DRG) and trigeminal ganglion, are specialized to transmit sensory information from the periphery to the central nervous system. Our molecular understanding of

    ...
    Optical Clearing and 3D Analysis Optimized for Mouse and Human Pancreata

    The pancreas is a heavily innervated organ, but pancreatic innervation can be challenging to comprehensively assess using conventional histological methods. However, recent advances in whole-mount tissue clearing and 3D rendering techniques have

    ...
    Isolation and in vitro Culture of Mouse Oocytes
    Authors:  Jessica Greaney, Goutham Narayanan Subramanian, Yunan Ye and Hayden Homer, date: 08/05/2021, view: 23, Q&A: 0

    Females are endowed at birth with a fixed reserve of oocytes, which declines both in quantity and quality with advancing age. Understanding the molecular mechanisms regulating oocyte quality is crucial for improving the chances of pregnancy success

    ...
    Efficient and Rapid Analysis of Polysomes and Ribosomal Subunits in Cells and Tissues Using Ribo Mega-SEC

    Polysome profile analysis is a popular method for separating polysomes and ribosomal subunits and is typically achieved using a sucrose density gradient (SDG). This has remained the gold standard method since ribosomes were first discovered;

    ...
    Mouse Periosteal Cell Culture, in vitro Differentiation, and in vivo Transplantation in Tibial Fractures

    The periosteum covering the outer surface of bone contains skeletal stem/progenitor cells that can efficiently form cartilage and bone during bone repair. Several methods have been described to isolate periosteal cells based on bone scraping and/or

    ...
    A Simple Spatial-independent Associative and Reversal Learning Task in Mice
    Authors:  Fulvio Magara, Benjamin Boury-Jamot and Hanna Hörnberg, date: 08/05/2021, view: 17, Q&A: 0

    The ability to adapt one's behavior in response to changing circumstances, or cognitive flexibility, is often altered in neuropsychiatric and neurodevelopmental conditions. In rodents, cognitive flexibility is frequently assessed using associative

    ...
    Cell-attached and Whole-cell Patch-clamp Recordings of Dopamine Neurons in the Substantia Nigra Pars Compacta of Mouse Brain Slices
    Authors:  Stefano Cattaneo, Maria Regoni, Jenny Sassone and Stefano Taverna, date: 08/05/2021, view: 17, Q&A: 0

    The Substantia Nigra pars compacta (SNc) is a midbrain dopaminergic nucleus that plays a key role in modulating motor and cognitive functions. It is crucially involved in several disorders, particularly Parkinson’s disease, which is

    ...
    Purification of Mitochondrial Ribosomes with the Translocase Oxa1L from HEK Cells
    Authors:  Hanting Yang and Nirupa Desai, date: 08/05/2021, view: 44, Q&A: 0

    Mitochondrial ribosomes (mitoribosomes) perform protein synthesis inside mitochondria, the organelles responsible for energy conversion and adenosine triphosphate (ATP) production in eukaryotic cells. To investigate their functions and structures,

    ...
    Purification and Cryo-electron Microscopy Analysis of Plant Mitochondrial Ribosomes
    Authors:  Florent Waltz, Philippe Giegé and Yaser Hashem, date: 08/05/2021, view: 19, Q&A: 0

    Plants make up by far the largest part of biomass on Earth. They are the primary source of food and the basis of most drugs used for medicinal purposes. Similarly to all eukaryotes, plant cells also use mitochondria for energy production. Among

    ...
    Purification of Recombinant Wild Type and Mutant Ryanodine Receptors Expressed in HEK293 Cells

    High quantities of purified ryanodine receptor (RyR), a large (2.26 MDa) intracellular homotetrameric membrane protein, can be obtained from heterologous expression in HEK293 cells and used for structure determination by cryo-EM. The advantage of

    ...
    Fe-NTA Microcolumn Purification of Phosphopeptides from Immunoprecipitation (IP) Eluates for Mass Spectrometry Analysis
    Authors:  Ethan J. Sanford and Marcus B. Smolka, date: 08/05/2021, view: 14, Q&A: 0

    Protein phosphorylation is a nearly universal signaling mechanism. To date, a number of proteomics tools have been developed to analyze phosphorylation. Phosphoproteome-wide analyses using whole cell extracts suffer from incomplete coverage, often

    ...
    A Genetically Engineered Mouse Model of Venous Anomaly and Retinal Angioma-like Vascular Malformation
    Authors:  Xudong Cao, Beibei Xu, Xiao Li, Taotao Li and Yulong He, date: 08/05/2021, view: 16, Q&A: 0

    Characterization of key regulators in vein development will advance our understanding of mechanisms underlying venous anomalies and provide therapeutic targets for the treatment of vascular malformations. Here, we provide a detailed protocol for the

    ...
    Isolation of Nuclei from Mouse Dorsal Root Ganglia for Single-nucleus Genomics
    Authors:  Lite Yang, Ivan Tochitsky, Clifford J. Woolf and William Renthal, date: 08/05/2021, view: 24, Q&A: 0
    [Abstract]

    Primary somatosensory neurons, whose cell bodies reside in the dorsal root ganglion (DRG) and trigeminal ganglion, are specialized to transmit sensory information from the periphery to the central nervous system. Our molecular understanding of peripheral sensory neurons has been limited by both their heterogeneity and low abundance compared with

    ...
    Optical Clearing and 3D Analysis Optimized for Mouse and Human Pancreata
    [Abstract]

    The pancreas is a heavily innervated organ, but pancreatic innervation can be challenging to comprehensively assess using conventional histological methods. However, recent advances in whole-mount tissue clearing and 3D rendering techniques have allowed detailed reconstructions of pancreatic innervation. Optical clearing is used to enhance tissue

    ...
    Isolation and in vitro Culture of Mouse Oocytes
    Authors:  Jessica Greaney, Goutham Narayanan Subramanian, Yunan Ye and Hayden Homer, date: 08/05/2021, view: 23, Q&A: 0
    [Abstract]

    Females are endowed at birth with a fixed reserve of oocytes, which declines both in quantity and quality with advancing age. Understanding the molecular mechanisms regulating oocyte quality is crucial for improving the chances of pregnancy success in fertility clinics. In vitro culture systems enable researchers to analyse important molecular and

    ...
    Efficient and Rapid Analysis of Polysomes and Ribosomal Subunits in Cells and Tissues Using Ribo Mega-SEC
    [Abstract]

    Polysome profile analysis is a popular method for separating polysomes and ribosomal subunits and is typically achieved using a sucrose density gradient (SDG). This has remained the gold standard method since ribosomes were first discovered; however, this method is time-consuming and requires multiple steps from making the gradient and long

    ...
    Mouse Periosteal Cell Culture, in vitro Differentiation, and in vivo Transplantation in Tibial Fractures
    [Abstract]

    The periosteum covering the outer surface of bone contains skeletal stem/progenitor cells that can efficiently form cartilage and bone during bone repair. Several methods have been described to isolate periosteal cells based on bone scraping and/or enzymatic digestion. Here, we describe an explant culture method to isolate periosteum-derived

    ...
    A Simple Spatial-independent Associative and Reversal Learning Task in Mice
    Authors:  Fulvio Magara, Benjamin Boury-Jamot and Hanna Hörnberg, date: 08/05/2021, view: 17, Q&A: 0
    [Abstract]

    The ability to adapt one's behavior in response to changing circumstances, or cognitive flexibility, is often altered in neuropsychiatric and neurodevelopmental conditions. In rodents, cognitive flexibility is frequently assessed using associative learning paradigms with a reversal component. The majority of existing protocols rely on

    ...
    Cell-attached and Whole-cell Patch-clamp Recordings of Dopamine Neurons in the Substantia Nigra Pars Compacta of Mouse Brain Slices
    Authors:  Stefano Cattaneo, Maria Regoni, Jenny Sassone and Stefano Taverna, date: 08/05/2021, view: 17, Q&A: 0
    [Abstract]

    The Substantia Nigra pars compacta (SNc) is a midbrain dopaminergic nucleus that plays a key role in modulating motor and cognitive functions. It is crucially involved in several disorders, particularly Parkinson’s disease, which is characterized by a progressive loss of SNc dopaminergic cells. Electrophysiological studies on SNc neurons are

    ...
    Purification of Mitochondrial Ribosomes with the Translocase Oxa1L from HEK Cells
    Authors:  Hanting Yang and Nirupa Desai, date: 08/05/2021, view: 44, Q&A: 0
    [Abstract]

    Mitochondrial ribosomes (mitoribosomes) perform protein synthesis inside mitochondria, the organelles responsible for energy conversion and adenosine triphosphate (ATP) production in eukaryotic cells. To investigate their functions and structures, large-scale purification of intact mitoribosomes from mitochondria-rich animal tissues or HEK cells

    ...
    Purification and Cryo-electron Microscopy Analysis of Plant Mitochondrial Ribosomes
    Authors:  Florent Waltz, Philippe Giegé and Yaser Hashem, date: 08/05/2021, view: 19, Q&A: 0
    [Abstract]

    Plants make up by far the largest part of biomass on Earth. They are the primary source of food and the basis of most drugs used for medicinal purposes. Similarly to all eukaryotes, plant cells also use mitochondria for energy production. Among mitochondrial gene expression processes, translation is the least understood; although, recent advances

    ...
    Purification of Recombinant Wild Type and Mutant Ryanodine Receptors Expressed in HEK293 Cells
    [Abstract]

    High quantities of purified ryanodine receptor (RyR), a large (2.26 MDa) intracellular homotetrameric membrane protein, can be obtained from heterologous expression in HEK293 cells and used for structure determination by cryo-EM. The advantage of using recombinant protein is that the variability due to post-translational modifications can be

    ...



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