Protocols for In Situ Hyb:
Detailed worksheet (HTML | PDF | Word)
Short worksheet (HTML | PDF | Word)
Short Worksheet FAST protocol (HTML | PDF | Word)
Protocols for Making Antisense RNA probe (dig labelled)
Long (HTML | Download PDF)
Short (HTML | Download PDF)
The X. laevis procedure for whole mount in situ hybridization works perfectly well for X. tropicalis embryos without change. We use the protocol by Sive et al. Early Development of Xenopus laevis: A Laboratory Manual CSHL Press (2000).
In order to efficiently perform the procedure we routinely place embryos in porous baskets to facilitate the solution exchanges. Additionally we use the following worksheets during the procedure.
For those new to the procedure, the entire procedure is outlined in detail in this worksheet (PDF | Word).
For those more experienced with the procedure, the entire protocol is summarized in this short two page worksheet (PDF| Word).
Standard Protocol Modifications
In an attempt to streamline the procedure, we have compared the original protocol to a modified protocol with particular steps omitted. In a side by side comparison, omission of the proteinase K step led to a signifcant reduction in signal especially in neural and endodermal staining.
The first day of the in situ protocol is considerably prolonged because of the long pre-hybridization step (six hours). We have tested the minimum length of time required for pre-hybridization. In X. laevis embryos, the prehybridization step may be reduced from six to four hours but must last for at least four hours. Shorter pre-hyb times led to a degradation in signal.
Using a subset of probes we have found that in X. tropicalis embryos the pre-hybridization step appears unecessary and equivalent signal can be obtained if the pre-hybridization is omitted, reduced to one hour, or kept at the full six hours. Eliminating the pre-hybridization step greatly shortens the first day of the protocol. Of course only a limited number of probes were tried, and we recommend comparing the standard procedure with and without pre-hybridization to be sure that the signal is equivalent. In routine practice, we pre-hyb for one hour at a minimum which works well and is convenient.
Shorten MAB washes
Additionally we have shortened the MAB washes after the antibody step. The original procedure called for multiple 1-2 hour washes. Instead we have shortened this to two 5 min washes followed with an overnight wash at 4oC. Then in the morning we do an additional three washes for 5 to 15 minutes each in MAB before the AP buffer step. This modification has also greatly sped up the in situ process. We can now complete the entire procedure in as short as three days with embryos ready for photography. These modifications are made in the following protocol:
Short Worksheet without pre-hyb and quick MAB. (PDF | Word)
Shorter Antibody Incubation
We have also tested shortening the anti-dig antibody incubation step from four hours at room temp to just one hour. This however did seem to result in a degradation of signal. Extremely robust probes may still give sufficient signal, but four hours of antibody incubation does appear essential.
Making Antisense Probes for in situ
Like our in situ worksheets, we have created two probe worksheets. The longer probe worksheet gives a great deal of detail and is useful for beginners. The short worksheet (PDF) is summarized on a single page for the advanced user.
These protocols were written in summary form by Joanna Yeh and Mustafa Khokha
Modifications of in situ protocol tested by Joanna Yeh and Lisa Gaw