{"EMPIAR-12513":{"imagesets":[{"segmentations":[],"name":"Volume electron microscopy of APEX2-DAB labeled thalamocortical projections in visual cortex L4","directory":"data/shared_data","category":"reconstructed volumes","header_format":"TIFF","data_format":"TIFF","num_images_or_tilt_series":4,"frames_per_image":1,"frame_range_min":null,"frame_range_max":null,"voxel_type":"UNSIGNED BYTE","pixel_width":null,"pixel_height":null,"micrographs_file_pattern":"","picked_particles_file_pattern":"","picked_particles_directory":"","details":"","image_width":null,"image_height":null}],"workflow_file":null,"grant_references":[{"funding_body":"National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)","code":"R01/NS089791","country":"United States"}],"version_history":[],"title":"Volume electron microscopy of APEX2-DAB labeled thalamocortical projections in visual cortex L4","principal_investigator":[{"author_orcid":"0000-0002-7542-5930","middle_name":"J","organization":"Salk Institute for Biological Studies","street":"10010 North Torrey Pines Rd","town_or_city":"La Jolla","state_or_province":"California","post_or_zip":"92037","telephone":"+1 858 453 4100","fax":null,"first_name":"Nicola","last_name":"Allen","email":"nallen [at] salk.edu","country":"United States","entry":"EMPIAR-12513"}],"status":"REL","deposition_date":"2024-11-20","release_date":"2025-01-29","obsolete_date":null,"update_date":"2025-01-29","corresponding_author":{"author":{"author_orcid":"0000-0002-7542-5930","middle_name":"J","organization":"Salk Institute for Biological Studies","street":"10010 North Torrey Pines Rd","town_or_city":"La Jolla","state_or_province":"California","post_or_zip":"92037","first_name":"Nicola","last_name":"Allen","country":"United States"}},"authors":[{"author":{"name":"Bosworth AP","author_orcid":null}},{"author":{"name":"Weiser Novak S","author_orcid":"0000-0002-8344-9772"}},{"author":{"name":"Manor U","author_orcid":"0000-0002-9802-1955"}},{"author":{"name":"Allen NJ","author_orcid":"0000-0002-7542-5930"}}],"cross_references":[],"biostudies_references":[],"idr_references":[],"empiar_references":[],"citation":[{"authors":[{"name":"Bosworth A","author_orcid":null},{"name":"Contreras M","author_orcid":null},{"name":"Weiser Novak S","author_orcid":null},{"name":"Sancho L","author_orcid":null},{"name":"Salas I","author_orcid":null},{"name":"Manor U","author_orcid":"0000-0002-9802-1955"},{"name":"Allen N","author_orcid":null}],"editors":[],"published":true,"j_or_nj_citation":true,"title":"Astrocyte glypican 5 regulates synapse maturation and stabilization","volume":null,"country":"","first_page":null,"last_page":null,"year":"2023","language":"English","doi":"10.1101/2023.03.02.529949","pubmedid":null,"details":"Adapted from DOI: 10.1101/2023.03.02.529949\n\nTo label thalamocortical projections for EM reconstruction, AAV9-COX4-dAPEX2 was injected into the dLGN of WT and cKO mice at P14 (2 littermate pairs). pAAV-COX4-dAPEX2 was a gift from David Ginty (Addgene plasmid #117176; http://n2t.net/addgene:117176; RRID:Addgene_117176). Packaging in AAV9 was performed by the Salk Viral Vector core facility (GT3) at a concentration of 2×1014 vg/mL. Mice were anesthetized with oxygenated isoflurane (2-3%) and injection was done with a Nanoject pressure injection system. Virus was diluted to 3×1012 vg/mL and injected at coordinates 2.0 mm posterior from Bregma, 1.9 mm lateral from the midline, and 2.9 mm below the pia, with a total of 150nL of virus delivered at a rate of 2nL per second. Following 2 weeks of expression, mice were collected at P28 as described in the methods section of the paper and the brain was processed for electron microscopy. Materials used for processing samples for EM were sourced from Electron Microscopy Sciences unless otherwise indicated. All steps were performed at ice cold temperatures unless otherwise indicated.\n\nBrains were mounted on a Leica VT1000S vibrating microtome in cacodylate buffer, and 100µm coronal sections containing the primary VC collected in 6 well plates and washed 2×10 minutes in cacodylate buffer supplemented with 50mM glycine, followed by 1×10 minutes in cacodylate buffer. A 10X diaminobenzidine (DAB) tetrahydrochloride solution was freshly prepared by dissolving 50mg of DAB in 0.1 M HCl at room temperature prior to tissue processing. Sections were then incubated in DAB solution (final concentration of 0.3 mg/mL DAB in cacodylate buffer) for 30 minutes in the dark. After 30 minutes, 10µL/mL of cacodylate supplemented with 0.3% H2O2 was added directly to the DAB solution (final H2O2 concentration of 0.003%) and swirled extensively to initiate the peroxidase reaction which was allowed to proceed for 1 hour in the dark. Slices were evaluated for reaction product and washed 3×10 minutes in cacodylate buffer and then further post-fixed overnight in cacodylate buffer with 3% glutaraldehyde.\n\nThe following day sections were rinsed 2×10 minutes in cacodylate buffer with 50mM glycine followed by 1×10 minutes in cacodylate buffer and transferred to a petri dish filled with ice cold cacodylate buffer for photography and microdissection. 2mm wide strips spanning from the cortical surface to the corpus collosum were collected into scintillation vials for further processing. Samples were stained with reduced osmium (1% osmium tetroxide and 1.5% potassium ferrocyanide in cacodylate buffer) for 1h at room temperature, then rinsed 5x3 minutes with ice cold water and left in 1% aqueous uranyl acetate at 4°C overnight. Samples were then serially dehydrated in ice cold aqueous ethanol solutions of ascending concentrations, before 3×10 minute incubations with absolute ethanol at room temperature. Samples were then infiltrated with ascending concentrations of Durcupan resin in absolute ethanol at room temperature (3:1, 4h; 1:1, 4h; 1:3, overnight) before 2x4h incubations rotating in pure resin. Samples were embedded with fresh resin and paper labels in silicon molds, with the tissue oriented en face to the block face and polymerized for 60h at 65°C in an oven.\n\nSerial sections were collected onto silicon wafers. Briefly, the block was trimmed using a 90° diamond trimming knife (Diatome) on an ultramicrotome (Leica UC7) to a trapezoidal frustum of roughly 150×400µm which included the region from the cortical surface to deep cortical layers. A silicon chip (35x7mm; University Wafer, Boston, MA) was hydrophilized in a plasma cleaner (Harrick) immediately preceding partial immersion in the water boat of a Histo knife (Diatome) mounted on the ultramicrotome. Ribbons of 150-200 serial sections of thicknesses of approximately 55nm were cut with 4 drops of pure ethanol in the water boat and an ionizing instrument (Leica EM Crion) activated and oriented towards the cutting edge of the knife from above. When ribbons of sufficient quality and length were generated, they were released from the knife edge using a single-eyelash brush and carefully positioned over the chip. The water level was then slowly lowered using a peristaltic pump, and sections were allowed to dry down on the silicon substrate. Chips were briefly dried on a slide on a hot plate set to 60°C.\n\nChips were mounted on aluminum stubs using carbon sticky tabs and loaded into a scanning electron microscope (SEM; Zeiss Sigma VP) equipped with a sensitive backscatter detector (Gatan), as well as extended raster scanning capabilities and a control system designed for serial section imaging workflows (ATLAS5, FIBICS). Low resolution image maps of the ribbon of serial sections were collected, and a mid-resolution map of a central section was generated for reference. From this image, a region of interest (ROI) from VC L4 of 50×50µm was selected from between 250-350µm from the cortical surface that [1] had DAB+ terminals; [2] was not obstructed by blood vessels or somata; [3] was free from obvious debris throughout the series as assessed from the low-resolution map. This region was identified at one end of the ribbon of sections, and the ROI was imaged at high resolution (pixel size: 2nm; dwell time: 6µs; EHT: 3kV; aperture: 30µm; working distance: 8-9mm) on every section in the ribbon.\n\nImage stacks were collated and rigidly aligned using TrakEM2 in Fiji and cropped to a minimum continuous cube of roughly aligned data with minimal padding. Fine stack alignment was accomplished using SWiFT-IR as deployed on 3DEM.org using the TACC compute resource Stampede 2.","book_chapter_title":null,"publisher":null,"publication_location":null,"journal":"bioRxiv","journal_abbreviation":"","issue":null,"preprint":true}],"dataset_size":"199.8 GB","experiment_type":"ssET","scale":"cell","entry_doi":"10.6019/EMPIAR-12513"}}