3D Gene Expression

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Overview:
   Summary
   Project Goals

Methods:
   Staining & Mounting
   Probe Constructs
   Staging
   Imaging

Software:
   PointCloudXplore
   PointCloudToolBox
   PointCloudAlign
   NODE

File Formats:
   PointCloud (.pce)
   VirtualEmbryo (.vpc)
   Correspondence files

Staining and Mounting

This page contains links to detailed protocols for collecting, fixing, staining, and mounting embryos and for making probes for in situ-hybridizations. Information on plasmid DNA constructs is also provided.
We stain embryos to detect nuclei and the expression patterns of either two mRNAs, two proteins, or a protein and an mRNA.

Embryo collection
Embryos of the required range of developmental stages are fixed with formaldehyde and then stored in -20°C [Collection and Fixation Protocol].

Staining for mRNA expression
To detect mRNAs, antisense RNA probes are transcribed in vitro from PCR-products [RNA Probe Protocol]. Information on the DNA probe constructs used is available. Each probe is labeled with either digoxigenin (DIG), dinitrophenyl (DNP), fluorescien-FTIC or biotin (BIO) - UTP. Two differently labeled probes are then hybridized to embryos. Each probe is detected sequentially using a horseradish peroxidase (HRP) conjugated antibody against the probe label and a tyramide linked to a fluor (mRNA Detection Protocol). The tyramide linked fluors we use are coumarin and Cy3. In the end, nuclei are labeled with the nucleic acid binding dye Sytox Green.

Staining for mRNA and protein expression
Embryos are hybridized with a nucleic acid probe as above. The labeled probe is detected using the HRP- conjugated antibody and coumarin-tyramide. At the same time, the protein of interest is labeled with primary antibody against it and the nuclei are labeled either using an antibody against the nuclear protein histone H1 or using Sytox Green dye. The proteins are then detected using an AlexaFluor-conjugated secondary antibody [mRNA/Protein Detection Protocol].

Staining for protein expression
To detect protein expression, specific antibodies and differently conjugated AlexaFluor-secondary antibodies are used. Nuclei are labeled using an antibody against the nuclear protein histone H1 or Sytox Green.

Mounting
Embryos stained by any of the above methods are mounted in DePex using coverslip bridges to protect the embryos from flattening. This mountant closely matches the refractive index of the embryo allowing us to image all the way through and protects the fluors from bleaching.