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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used to prepare Phosphate Buffered Saline (PBS). PBS (-) and PBS (+) which includes Tween are used as biologically neutral wash solutions in many processes including In Situ Hybridization (ISH), Immunohistochemistry (IHC) and Fixation.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. Phosphate Buffered Saline (PBS). protocols.io https://dx.doi.org/10.17504/protocols.io.bezgjf3w Copy   

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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.

Proper citation: Allen Institute for Brain Science 2020. PCR Prep from cDNA for IVT Reactions. protocols.io https://dx.doi.org/10.17504/protocols.io.bcsfiwbn 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 2020. Mouse Whole Cell Tissue Processing for 10x Genomics Platform. protocols.io https://dx.doi.org/10.17504/protocols.io.be2fjgbn Copy   

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Authors: ZengU19 BRAIN Grant
Group: BICCN, Allen Institute for Brain Science
Summary: Protocol to generate Nextera libraries using 0.2x reagents in a 96-well PCR plate using the Formulatrix Mantis instrument.

Proper citation: ZengU19 BRAIN Grant 2019. Nextera XT at 0.2X On the Mantis. protocols.io https://dx.doi.org/10.17504/protocols.io.63rhgm6 Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used to produce crude preps of AAV of any serotype.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. Production of Crude AAV Virus Extract. protocols.io https://dx.doi.org/10.17504/protocols.io.bdvwi67e Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol outlines strategies to solve common problems that occur when setting up the TissueCyte 1000 system for normal operation.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. TissueCyte Troubleshooting Guide. protocols.io https://dx.doi.org/10.17504/protocols.io.beryjd7w Copy   

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Authors: ZengU19 BRAIN grant
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the procedure for transcardial perfusion of an adult mouse, followed by brain dissection, embedding, slicing, blockface imaging, ROI microdissection, and tissue dissociation. The output of this protocol is a cell suspension that can be sorted via FACS for single-cell transcriptional profiling.

Proper citation: ZengU19 BRAIN grant 2019. Slice Preparation with Tissue Dissociation - Mouse Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7dwhi7e 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.bd8vi9w6 Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the process to obtain electrophysiological recordings and cellular contents from neurons in postnatal mouse and/or human brain slices.

Proper citation: Allen Institute for Brain Science 2020. Patch-Seq Recording and Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.bcthiwj6 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 a fixed mouse brain specimen bonded to an agarose block for blockface imaging and sectioning using the TissueCyte 1000 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. TissueCyte Specimen Embedding Acrylamide Coembedding. protocols.io https://dx.doi.org/10.17504/protocols.io.bdpvi5n6 Copy   

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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 https://dx.doi.org/10.17504/protocols.io.be2cjgaw Copy   

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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.

Proper citation: Allen Institute for Brain Science 2020. DAB Detection of Biocytin Labeled Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.bctbiwin Copy   

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Authors: LeinU01 BRAIN Grant
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the slicing and microdissection procedure on frozen post-mortem human brain tissue to be used to isolate nuclei for subsequent transcriptional profiling.

Proper citation: LeinU01 BRAIN Grant 2019. Human Tissue Slicing and Dissections for Nuclear Isolations. protocols.io https://dx.doi.org/10.17504/protocols.io.7aehibe Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: Protocol to generate Nextera libraries using 0.2x reagents in a 96-well PCR plate using the Formulatrix Mantis instrument.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. Nextera XT at 0.2X On the Mantis. protocols.io https://dx.doi.org/10.17504/protocols.io.brdjm24n 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.

Proper citation: Allen Institute for Brain Science 2020. Mouse Whole Cell Tissue Processing for 10x Genomics Platform. protocols.io https://dx.doi.org/10.17504/protocols.io.bc6bizan Copy   

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Authors: Carly Martin, Abdul Abdul, Charles Vanderburg, Naeem Nadaf, Ashley Feirrera, Evan Macosko
Group: BICCN
Summary: Protocol for extraction of nuclei from frozen tissue in preparation for single-nuclei sequencing (droplet-based/10X). This protocol is based strongly on a similar extraction protocol from the McCarroll lab.

Proper citation: Carly Martin, Abdul Abdul, Charles Vanderburg, Naeem Nadaf, Ashley Feirrera, Evan Macosko 2020. Frozen Tissue Nuclei Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.bbseinbe Copy   

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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 2021. Tris Buffered Saline (TNT). protocols.io https://dx.doi.org/10.17504/protocols.io.brdkm24w Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: The protocol describes how 1x3 glass microscope slides are coated with gelatin to improve adherence of fixed adult mouse brain sections or other tissue to 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. Coating Slides with Gelatin. protocols.io https://dx.doi.org/10.17504/protocols.io.bf64jrgw 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.bg52jy8e 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.bepzjdp6 Copy   

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