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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 https://dx.doi.org/10.17504/protocols.io.bd8qi9vw Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the procedures for intracardiac perfusion fixation of postnatal mice, including anesthesia, exsanguination, fixation, brain removal and post-fixation storage. 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. Mouse Cardiac Perfusion Fixation and Brain Collection. protocols.io https://dx.doi.org/10.17504/protocols.io.be2djga6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.y6nfzde Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: Isolation of nuclei from frozen adult human brain tissue or thawed and microdissected brain tissue sections for RNA-seq analysis. Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U01MH114812. 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. Isolation of Nuclei from Adult Human Brain Tissue for 10x Genomics Platform. protocols.io https://dx.doi.org/10.17504/protocols.io.bq64mzgw Copy   

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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 https://dx.doi.org/10.17504/protocols.io.besbjean Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: 50 mM Phosphate Buffer can be used as a general buffer solution for multiple applications. It is suitable for use as a buffer for specimen embedding, as part of brain preparation associated with TissueCyte 2P Serial Imaging, described in PF0315 TissueCyte Specimen Embedding Acrylamide Coembedding.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. 50 mM Phosphate Buffer (with 0.02% sodium azide option). protocols.io https://dx.doi.org/10.17504/protocols.io.bezjjf4n Copy   

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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 https://dx.doi.org/10.17504/protocols.io.bdpfi5jn Copy   

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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 https://dx.doi.org/10.17504/protocols.io.bg4wjyxe Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the method for preparing PBS with 0.1% Sodium Azide for storage and/or processing of fixed 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. PBS with 0.1% Sodium Azide. protocols.io https://dx.doi.org/10.17504/protocols.io.bfzsjp6e Copy   

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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 https://dx.doi.org/10.17504/protocols.io.bezdjf26 Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used for the PCR reaction from clone templates in glycerol stocks generating transcript templates ready for IVT reaction.Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number 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. PCR Prep from Glycerol Stock for IVT Reactions. protocols.io https://dx.doi.org/10.17504/protocols.io.brt2m6qe Copy   

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Authors: Sebastian Preissl, Xinxin Wang, Bing Ren
Group: BICCN
Summary: Protocol for applying the TN5-based ATAC-seq method to the analysis of the chromatin of single cell nuclei isolated from brain tissue, as performed within the Center for Epigenomics of the Mouse Bain Atlas (CEMBA) and in coordination with the BRAIN Initiative Cell Census Network (BICCN).

Proper citation: Sebastian Preissl, Xinxin Wang, Bing Ren 2018. Sequencing open chromatin of single cell nuclei: snATAC-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.pjudknw Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the instructions for setup and scanning of fluorescently-labeled tissue sections mounted in agarose and attached to a slide using the TissueCyte 1000 block face 2-photon system.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. Core Protocol for Serial Imaging of Fluorescently-Labeled Mouse Brain on the TissueCyte 1000 System. protocols.io https://dx.doi.org/10.17504/protocols.io.bdvmi646 Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the synthesis of first-strand cDNA from total RNA using the Invitrogen SuperScript III Reverse Transcriptase system.

Proper citation: Allen Institute for Brain Science 2019. cDNA Synthesis. protocols.io https://dx.doi.org/10.17504/protocols.io.9geh3te Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This SOP provides instructions to prepare Internal Solution II used for simultaneous patch clamp electrophysiology experiments to probe circuit connectivity.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. Electrode Internal Solution with Biocytin II. protocols.io https://dx.doi.org/10.17504/protocols.io.bepnjdme Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes how to isolate tissue from various ROI’s of the mouse brain followed by preparation of a highly concentrated single cell suspension for transcriptomic profiling on 10x Genomics platform.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 2021. Mouse Whole Cell Tissue Processing for 10x Genomics Platform. protocols.io https://dx.doi.org/10.17504/protocols.io.bsfdnbi6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.bfzvjp66 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.bepsjdne Copy   

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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.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. Maintenance of TissueCyte System. protocols.io https://dx.doi.org/10.17504/protocols.io.ber3jd8n Copy   

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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 https://dx.doi.org/10.17504/protocols.io.bcseiwbe Copy   

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