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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocols provides detailed instructions for procedures needed for maintenance of the TissueCyte scanning systems.
Proper citation: Allen Institute for Brain Science 2020. Maintenance of TissueCyte System. protocols.io dx.doi.org/10.17504/protocols.io.bbx6ipre Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used to prepare 0.1M Phosphate Buffer. 0.1M Phosphate Buffer is used in the preparation of Sucrose solutions for cryoprotection.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. 0.1M Phosphate Buffer. protocols.io dx.doi.org/10.17504/protocols.io.bg43jyyn Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the delivery of a neuronal tracer using the Nanoject II. The surgery uses a stereotaxic system to target specific brain coordinates in the mouse. 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. Injection of Viral Tracers by Nanoject. protocols.io dx.doi.org/10.17504/protocols.io.bd8qi9vw Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used to prepare Tris Buffered Saline (TNT). 10X TNT is the stock solution used to prepare 1X TNT, which equilibrates tissue and maintains pH in physiological range.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. Tris Buffered Saline (TNT). protocols.io dx.doi.org/10.17504/protocols.io.bgncjvaw Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: Freshly prepared 4% Paraformaldehyde in PBS is used to fix adult and developing mouse brains during the transcardial perfusion process, as well as for immersion fixation. This solution can be used for no more than one week post-preparation date.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. Fresh 4% Paraformaldehyde in PBS. protocols.io dx.doi.org/10.17504/protocols.io.bezijf4e Copy
Authors: ZengU19 BICCN Grant
Group: BICCN, Allen Institute for Brain Science
Summary: We have developed a multiplexed single molecule FISH protocol for use at the Institute. This protocol was optimized on human tissue, but will work on mouse tissue as well. It was adapted from Lyubimova et. al., Nature Protocols, 2013.
Proper citation: ZengU19 BICCN Grant 2019. Sequential smFISH. protocols.io dx.doi.org/10.17504/protocols.io.y6nfzde Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the process for diaminobenzidine (DAB) detection of biocytin filled cells. This protocol is optimized for use with brain slices cut at 350 µm thick, in which cells are first filled with biocytin (i.e., post-electrophysiological recording), fixed in 4% PFA/2.5% glutaraldehyde, and transferred to PBS until ready to stain.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. DAB Detection of Biocytin Labeled Tissue. protocols.io dx.doi.org/10.17504/protocols.io.bep5jdq6 Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol provides step by step instructions for performing single-cell sorting by using BD FACSAria II machine or BD FACSAria Fusion machine and BD FACSDiva software.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. FACS Single Cell Sorting. protocols.io dx.doi.org/10.17504/protocols.io.besbjean Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes general procedures used for subcutaneous injection in adult mice.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. Subcutaneous Injection Adult Mouse. protocols.io dx.doi.org/10.17504/protocols.io.bezdjf26 Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: Protocol is used for the manufacture of glass pipettes for patch clamp electrophysiology using the DMZ-Zeitz Universal puller.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. Pulling Pipettes using the DMZ-Zeitz Universal. protocols.io dx.doi.org/10.17504/protocols.io.bdpfi5jn Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used for the degassing of critical reagents used in both the colorimetric and fluorescent in situ hybridization (ISH). This is crucial in eliminating hybridization bubbles from forming on the tissue. Hybridization bubbles result in no expression where they are located on the tissue.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. Degassing Reagents. protocols.io dx.doi.org/10.17504/protocols.io.bg4wjyxe Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used for the rapid generation of 3’ transcriptomic-NGS-ready- single-cell-libraries from pools of cells.Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number's 1U01MH114812-01 and 1U19MH114830-01. 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 2021. 10Xv3.1 Genomics Sample Processing. protocols.io dx.doi.org/10.17504/protocols.io.brt5m6q6 Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes bioanalyzer operation. The Agilent Bioanalyzer is used as a quality control step for PCR and IVT products. Peak size, shape, and sample concentration are recorded and used to evaluate samples on a pass/fail level.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. 2100 Bioanalyzer Operation for PCR, GTP and IVT Products. protocols.io dx.doi.org/10.17504/protocols.io.bfzjjp4n Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the process for diaminobenzidine (DAB) detection of biocytin filled cells. This protocol is optimized for use with brain slices cut at 350 µm thick, in which cells are first filled with biocytin (i.e., post-electrophysiological recording), fixed in 4% PFA/2.5% glutaraldehyde, and transferred to PBS until ready to stain.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. DAB Detection of Biocytin Labeled Tissue. protocols.io dx.doi.org/10.17504/protocols.io.bg5yjy7w Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the preparation of gelatin solution to be used for coating glass microscope slides.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. Preparation of Gelatin Slide Coating Solution. protocols.io dx.doi.org/10.17504/protocols.io.bfzvjp66 Copy
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 dx.doi.org/10.17504/protocols.io.bd93i98n Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes making Artificial Cerebrospinal Fluid IV (ACSF.IV). ACSF.IV is used for multiple applications including incubation of fresh mouse brain slices prior to electrophysiological recording.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. Artificial Cerebrospinal Fluid IV (ACSF.IV). protocols.io dx.doi.org/10.17504/protocols.io.bepsjdne Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes PCR1 and PCR2 reactions beginning with cDNA to generate transcript templates ready for IVT reaction. This protocol is written for 192 (2x96) reactions but can also be used for fewer or more reactions by adjusting the amount of materials and equipment appropriately.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. PCR Prep from cDNA for IVT Reactions. protocols.io dx.doi.org/10.17504/protocols.io.bep7jdrn Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This SOP provides instruction to prepare Internal Solution which is used for patch clamp electrophysiology, modified for cleanliness and mRNA capture and sustainability.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. Patch-Seq Internal Solution with Biocytin. protocols.io dx.doi.org/10.17504/protocols.io.bdvti66n Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used to generate accurate digital representations of neuron morphologies from a variety of brain regions and species. Each reconstruction captures the positions and thicknesses of the soma, dendrites and axon of a biocytin-filled cell within a slice of brain tissue. To generate the reconstruction we use an image stack containing ~200-700 serial 2D images that captures the full extent of the cell within the slice. We use the Vaa3D (Terafly) program with a Kazom’s Mozak user interface (Mozak for short) to visualize the 2D images in 3D. Once the stack is loaded in Mozak, our reconstruction is generated by placing nodes in 3D space. The placement of these nodes is dependent on the signal in the images. Our final output, an SWC text file (.swc format), contains many thousand rows. Each row contains a node ID, an x, y, z coordinate, radius value, neurite type, and parent node ID. After tracing is complete, we preform post processing to provide radius values, check for errors and consistency then the SWCs are uploaded into our Laboratory Information Management System.
Proper citation: Allen Institute for Brain Science 2020. 3D Reconstruction of Neurons in Vaa3D. protocols.io dx.doi.org/10.17504/protocols.io.bcseiwbe Copy
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