Spatial transcriptomics for profiling the tropism of viral vectors in tissues
yet remains challenging in 3D volumes. Combining USeqFISH with viral tools also offers an opportunity to integrate multimodal features, including morphology, physiology and genetics, of single cells in tissue, as we demonstrate here in NHP tissue. We believe that USeqFISH can bridge spatial transcriptomics and AAV engineering, contributing to advancing viral tools for targeted gene delivery and their broader use in basic and translational research.
We cultured and transfected HEK293T cells with pAAV-CMV-TET-VP3-UBC-tTA-T2A-eGFP-WPRE-hGHpA or pAAV-CAG-mNeonGreen-WPRE-hGHpA plasmid after 24–48 hours by using PEI-MAX . Three days after transfection, cells were fixed with 4% PFA for 10 minutes at room temperature and stored in 70% EtOH at −20 °C until used.
For 1-probe RCAHCR experiments, NIH3T3 cells were cultured in DMEM with 10% FBS. We coated glass bottom plates with 0.1 mg mlof laminin for 1 hour at 37 °C. We seeded NIH3T3 cells at low density , fixed the cells in ~12 hours with 4% PFA and stored in 70% EtOH at −20 °C until used.Detailed animal procedures until tissue recovery are available below. Once harvested, the brains were sliced with a vibratome to a thickness of 50 μm or 75 μm .
For tissue slices, we added a few modifications to the protocol. First, once rehydrated, the samples were kept in the PACT monomer solution at 4 °C, overnight. Next, we formed the PACT gel by purging the solution with Nfor 5–10 minutes and immediately incubating it at 37 °C for 2 hours. After aspirating the excess gel, we washed the samples with 2× SSC 3–4 times and cleared them in 8% SDS at 37 °C, overnight.
For sequential labeling, we detached hairpin assemblies and initiators from the amplicon using a two-step stripping method and added another set of initiators and hairpins for the next round. For two-step stripping, we added unique 10-nt toehold sequences to one of the hairpin pairs (Supplementary Table
Brasil Últimas Notícias, Brasil Manchetes
Similar News:Você também pode ler notícias semelhantes a esta que coletamos de outras fontes de notícias.
Illumina faces short-read rivals - Nature BiotechnologyNew sequencers are poised to disrupt Illumina’s dominance. Users will need to navigate the opportunities and challenges on offer in an increasingly crowded market.
Consulte Mais informação »
Web3 Infrastructure Startup Spatial Labs Raises $10M.spatial_labs, creator of a hardware platform for creating digital twins of consumer goods, has raised $10 million led by blockchaincap. BrandyBetz reports
Consulte Mais informação »
2024 Polestar 2 Gets New RWD Trim, 455-HP Dual-Motor Performance PackThe base front-wheel-drive Polestar 2 has been dropped entirely for 2024.
Consulte Mais informação »
Dual citizen of France and Canada who mailed ricin to President Trump pleads guilty | CNN PoliticsA dual citizen of France and Canada pleaded guilty on Wednesday to sending letters containing homemade ricin to then-President Donald Trump at the White House and eight Texas law enforcement officials.
Consulte Mais informação »
B&W updates PI7 earbuds with better battery life for the same pricePlus adaptive noise cancellation, dual-drivers, and aptX.
Consulte Mais informação »