3D Gene Expression
Staining & Mounting
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
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.
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.