Resources
Neural marker antibodies
All neurons = beta-tubulin from DSHB, clone E7 (mouse), cite us
Progenitors = PCNA from Life Technologies, clone PC10 (mouse), cite us and this
Excitatory Neurons = vGLUT1 from Abcam #ab77822, (rabbit), cite us
Inhibitory Neurons = vGAT1 from Novus Bio #NBP1-89802, (rabbit)
Astrocytes = GFAP from EMD Millipore #MAB360, (mouse), cite this
Microglia = isolectinB4-FITC from Sigma #L2895, (bacteria, has FITC), cite this
Genotyping Protocol
We use a CRISPR genotyping protocol to estimate sgRNA efficiencies. You can find our current genotyping protocol here. This is a genomic DNA extraction followed by a PCR that we send for Sanger sequencing and then upload for sequence deconvolution.
Whole-Mount Immunostaining Protocol
This is great for staining stage 44+ tadpoles (X. tropicalis). We use a high-throughput staining setup (see baskets, racks below), but our protocol also works great in Eppendorf tubes with manual washes. Please cite this and also this! For staining of earlier stage embryos/tadpoles or for X. laevis, you need to add some permeabilization steps— contact Helen for more info (helen.willsey@ucsf.edu).
3D printer files for embryo/tadpole stamps
These are great for positioning animals. Just stamp them into liquified agarose and let it set.
Tadpole stamp (X. tropicalis, stages 44-47)
Embryos stamp (X. tropicalis, stages 1-20)
Tadpole stamp (X. laevis, stages 44-47)
These were originally conceived of by Richard Harland and Marta Truchado-Garcia, though these files are our creations. Please cite this and this.
3D printer files for ISH/IF basket racks
These are great for high-throughput staining, and don't require a table saw!
Small rack (fits 24 baskets), for glass dish use #900170 from Wheaton Inc.
Medium rack (fits 30 baskets), for glass dish use #900203 from Wheaton Inc.
Large rack (fits 60 baskets), for glass dish use #49WF37 from Grainger
Making Baskets Protocol, we use 200um mesh for IF (Spectra Mesh #146487) and 100um mesh for ISH (Spectra Mesh #146488)
This concept was originally developed by Richard Harland and Karen Liu, though the 3D printer files are our creations. Please cite the basket protocol and our protocol.
Clonotator
Clonotator is a web-based platform made by the Jeremy Willsey lab that integrates several bioinformatics tools to facilitate efficient screening and annotation of cDNA construct sequences for use in molecular biological experiments. Clonotator automatically translates the nucleotide sequence of the construct into an amino acid sequence, aligns the predicted sequence to a reference database of protein sequences and identifies the best protein and isoform match, annotates any variants present in the construct, and incorporates disease-associated mutations and transcriptomic data to help the user assess suitability for a particular biological context (e.g., isoform affected by disease-associated variant, isoform expressed in tissue of interest).