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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.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.bgm9ju96 Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: 3, 3´ Diaminobenzidine (DAB) is a commonly used chromogen for histological staining. In the presence of peroxidase enzyme, DAB produces a brown precipitate that is insoluble in alcohol and xylene. Peroxidase catalyzes hydrogen peroxide oxidation of substrate DAB. This SOP provides instructions to prepare four different formulations of DAB. DAB for Immunohistochemical staining (IHC), DAB for Acetylcholinesterase (AChE) Staining, Nickel Enchanced DAB for Biocytin staining, and DAB for Biocytin staining.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 Solutions. protocols.io dx.doi.org/10.17504/protocols.io.betzjep6 Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol provides step by step instructions for shutting down the BD FACS Aria II and cleaning it of potentially hazardous materials.
Proper citation: Allen Institute for Brain Science 2019. BD FACS Aria II Shutdown. protocols.io dx.doi.org/10.17504/protocols.io.bameic3e Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the capture of 20X multi-channel images using human and mouse tissue.
Proper citation: Allen Institute for Brain Science 2020. Zeiss AxioImager Multi Channel 20X Image Capture. protocols.io dx.doi.org/10.17504/protocols.io.bcskiwcw Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: To characterize the orientation, laminar position, and anatomical location of in vitro single cells reconstructed in mouse and human brain slices, a 3-step process is used. Briefly, 20X brightfield and/or fluorescent images of DAPI (4',6-diamidino-2-phenylindole) are annotated to determine layer position and brain region, of biocytin-filled 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 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. 20X Anatomic and Layer Identification for Biocytin Filled Cells. protocols.io dx.doi.org/10.17504/protocols.io.bey9jfz6 Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol provides step by step instructions for shutting down the BD FACS Aria II and cleaning it of potentially hazardous materials.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. BD FACS Aria II Shutdown. protocols.io dx.doi.org/10.17504/protocols.io.bdvpi65n Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the process to fix recorded/filled cells from mouse and human brain slices with 4% PFA/2.5% Glutaraldehyde in PBS for future histochemical processing.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. Post Patch Clamp Slice Fixation. protocols.io dx.doi.org/10.17504/protocols.io.bg5tjy6n Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used for preparation of 5 µM DAPI cell staining solution that is applicable for staining adult mouse brain sections.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. 5 uM DAPI cell staining solution. protocols.io dx.doi.org/10.17504/protocols.io.bg47jyzn 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 and BD FACSDiva software in preparation for single-cell sorting.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. BD FACS Aria II Start-up and Calibration. protocols.io dx.doi.org/10.17504/protocols.io.bdvqi65w 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.be4cjgsw Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: Artificial Cerebrospinal Fluid IX (ACSF.IX) is used for applications including tissue bath solution during electrophysiological recording for probing synaptic connections under physiological conditions.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 IX (ACSF.IX). protocols.io dx.doi.org/10.17504/protocols.io.beppjdmn Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used for the large-scale production of EnvA-pseudotyped recombinant rabies virus.
Proper citation: Allen Institute for Brain Science 2019. Production of Recombinant EnvA Rabies Virus. protocols.io dx.doi.org/10.17504/protocols.io.8dnhs5e 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.besnjede Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the QC step that occurs after the week of TissueCyte runs, and prior to analysis.
Proper citation: Allen Institute for Brain Science 2020. TissueCyte Image QC. protocols.io dx.doi.org/10.17504/protocols.io.bbybipsn Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used to prepare Artificial Cerebrospinal Fluid I (ACSF.I). ACSF.I is used for applications including transcardial perfusion prior to fresh mouse brain slicing and as a bathing solution during fresh mouse brain slicing. 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 I (ACSF.I). protocols.io dx.doi.org/10.17504/protocols.io.bezkjf4w 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.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. 3D Reconstruction of Neurons in Vaa3D. protocols.io dx.doi.org/10.17504/protocols.io.bdppi5mn Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: Artificial Cerebrospinal Fluid III (ACSF.III) is used for applications including tissue bath solution during 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 III (ACSF.III). protocols.io dx.doi.org/10.17504/protocols.io.beptjdnn 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 iontophoretic method. 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. Stereotaxic Surgery for Delivery of Tracers by Iontophoresis. protocols.io dx.doi.org/10.17504/protocols.io.bgpvjvn6 Copy
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 dx.doi.org/10.17504/protocols.io.bdpui5nw Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the QC step that occurs after the week of TissueCyte runs, and prior to 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 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 Image QC. protocols.io dx.doi.org/10.17504/protocols.io.bdpri5m6 Copy
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