Whole mount in situ method
Malcolm Bennett, University of Warwick
The whole mount in situ method is derived from the Tautz
and Pfeifle (1989) paper (Chromosoma 98:81-85) which
described the non-radioactive localisation of hunchback mRNA
in Drosophila embryos. Ludevid et al (1992) have adapted the
method in a Plant Physiol. paper (100, 1633-1639) to suit
young Arabidopsis seedlings. Other groups, such as Kwart et al
(1993) (Plant J., 4, 993-1002), have reported its use in
localising differential gene expression. I would still recommend
performing in situs using sections as well, but this provides a
useful means to have a quick 'look and see' at your mRNAs
spatial expression pattern.
The Ludevid et al (1992) paper provides only brief details of
their modification to the Tautz and Pfeifle paper, so I've written
out details of my protocol. All washing steps are conducted at
room temperature (unless stated in the text) and samples are
attached to a rotating wheel to facilitate washing. All solutions
are DEPC-treated (except Tris containing ones) in case
riboprobes are used.
- 1. Germinate Arabidopsis seed on 1% agar plates containing
MS (l X strength) + 1% sucrose, pH 5.8. Place the plates
vertically so not to damage the roots on later transfer to
- 2. Take Arabidopsis seedlings in the range 3-8 days post
germination, carefully removing them from the plate into a
small eppendorf containing 500 microlitres PBS + 67mM EGTA +
6% Formaldehyde (PBS + E + F) and fixing for 25 minutes at RTP.
- 3. Remove the fixative and store the seedlings in 5OO
microlitres 100% ethanol for 24 days at RTP (I've left them
longer than this and they appeared to have worked: this step
will remove all of the pigmentation in the aerial parts)
- 4. Remove the ethanol and wash 3 X 5 minutes with 5OO
microlitres PBS + 0.1% Tween 20 (termed PBT) attaching the
tubes to a rotating wheel to aid washing.
- 5. Proteinase K treat the samples (using the proteinase at a 5O
micrograms/ml working concentration) in PBS for 3-5 minutes.
Remove the protease solution and replace with 5OO microlitres
2 mg/ml glycine in PBT, mix and leave for 2 minutes to stop
residual protease activity.
- 6. Wash the samples in PBT 2 X 5 minutes, then refix in PBS +
E + F for a further 20 minutes, followed by 3 X 10 minute
washes in PBT.
- 7. Start the prehybridisation by washing the seedlings in a
1:1 mix of PBT and hybridisation solution (termed HS which
consists of 5 X SSC, 50% Formamide, 5O micrograms/ml heparin,
0.1% Tween 20 and 1OO micrograms boiled salmon sperm DNA)
for 20 minutes.
- 8. Continue the prehybridisation in HS at 55C (we use a PCR
block) for 20-60 minutes, then remove the majority (i.e. 400
microlitres of the 5OO microlitres total) of the HS.
- 9. Add the DIG labelled probe to the hybridising samples and
leave overnight at 55C.
N.B. We have found that DIG-labelled DNA probes work best,
with respect to background and consistent staining patterns,
but other workers (cited above) have used DIG riboprobes
successfully. We added 2 microlitres of a heat denatured PCR
reaction (approximately equivalent to 5Ong of probe) into the
- 10. Samples are carefully washed following hybridisation,
initially in 500 microlitres of HS, then gradually equilibrated
back to PBT using a series of HS:PBT wash buffers (4:1, 3:2, 2:3,
1:4), then finally PBT. All washes are for a 20 minute period.
- 11. Once the seedlings are in PBT they are incubated with a
1:2000 dilution of the DIG antibody (Boerhinger) at 4C
N.B. To ensure that the DIG antibody does not give background
staining, a 1:10 dilution of the antibody is preabsorbed for a
few hours with the equivalent Arabidopsis tissue* in a PBT + 2%
BSA solution. * This tissue is fixed in PBS + 67 mM EGTA + 3%
Formaldehyde, then washed briefly in PBT prior to
preabsorption with the DIG antibody.
- 12. The seedlings are washed 4 X 20 minutes in PBT (to
remove non-specific antibody binding), then 3 X 5 minutes in
lOOmM NaCl, 5OmM MgCl2, lOOmM Tris pH 9.5, 0.1% Tween 20
and lmM Levamisole (an inhibitor of lysosomal phosphatases).
- 13. The seedlings are transferred to a well (in a 12 well
microtitre dish) containing lml of the latter solution
supplemented with 4.5 microlitres NBT and 3.5 microlitres X-
phosphate solutions (in Boerhinger DIG kit) and left to incubate
at room temperature. If the samples need longer than a few
hours to develop (AUXl needed overnight), leave them at 4-8 C
- 14. Visualise the tissue under a binocular microscope and
photograph. Light field doesn't do the staining justice, so I
prefer to use dark-field, which also brings out the root