In Situ Hybridization

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. Links to the detailed and short protocols, as well as links for making RNA probe:

Protocols for In Situ Hyb:

Detailed worksheet (HTML | Word)
Short worksheet (HTML | Word)
Short Worksheet FAST protocol (HTML | Word)

Protocols for in Situ Robot: (HTML)

Protocol for Making Antisense RNA probe (dig labelled)

Standard Protocol Modifications

Eliminate PK

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 significant reduction in signal especially in neural and endodermal staining.

Eliminate Pre-hybridization

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 unnecessary 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.

These protocols were written by Mustafa Khokha