Double in situ hybridization is performed by hybridizing two differently labeled probes (digoxigenin and fluorescein labeled) at the same time. After the 1st antibody is used to visualize a probe, it is inactivated and then 2nd antibody and fluorescent substrate are used. Although two different antibodies are used, they both have the same enzyme conjugated.

For fluorescent substrates, we are using the TSA fluorescence systems from NEN/Perkin Elmer. Here is a scheme to describe the different steps of the procedures. For weaker probes (or lower amount of transcripts), we are using an additional amplification system (right column): (see below protocol for schematic, adapted from NEN/Perkin Elmer catalogue.)
We have used this protocol for embryos between 8-cell stage to 5.5 dpc but can probably be adapted for earlier or later stages.
All procedures are done in 4-well plates under a dissecting microscope. When necessary, embryos are transferred with a mouth pipet (pulled Pasteur pipet). All the procedures are carried out by carefully emptying the well, leaving the embryos in and pipetting in the new buffer (unless otherwise stated). These small embryos do not really need any agitation but use of a "nutator" will move them towards the center of the plate, making it easier to exchange the buffer.
All steps are at room temperature unless otherwise noted.

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1. Dissect or flush embryos in PBS or M2 (Nag yet al.,2003, Manipulating the mouse embryo, Cold Spring Harbor), then fix in 4% paraformaldehyde, for 1 h. (RT) to overnight (+4° C). It is not necessary to remove the Zonal Pellucid
2. Wash in PBT (PBS, 0.1% Tween 20), 2 times for 5 min.
3. Dehydrate through 25%, 50%, 75% methanol/PBT and twice 100% methanol, 5 min. for each step. Embryos are stored in 100% methanol at -20¾ C in the 4-well plate sealed with parafilm (evaporation can still happen and it is advisable to use the embryos relatively rapidly or to regularly add methanol 100%).

4. Rehydrate the embryos through 75%, 50%, 25% methanol in PBT and twice in PBT, for 5 min. at each step.
5. Permeabilise the embryos for 10 min, RT with RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% Na deoxycolate, 0.1% SDS, 1 mM EDTA, 50 mM Tris pH 8.0).
6. Wash 3 times for 5 min with PBT.
7. Fix in freshly prepared 0.2% glutaraldehyde+4% paraformaldehyde in PBS for 20 min.
8. Wash in PBT, twice 5 min.
9. Prehybridize at least 1 h at 65° C with hybridization buffer (50% formamide, 5X SSC, 1% SDS, 100 mg/ml yeast tRNA, 50 mg/ml heparin (50 mg/ml stock in 4X SSC).
10. Replace with hybridization buffer containing about 1 mg/ml digoxygenin- and/or fluorescein-labelled RNA probes. Incubate at 65° C overnight.
11. Wash 2 times for 30 min at 65° C with: 50% formamide, 5X SSC, 1% SDS.
12. Wash once 20 min at 65° C with 50% formamide, 2X SSC, 1% SDS.
13. Wash 2 times for 5 min with PBT or TNT (0.1M Tris pH 7.5; 0.15 M NaCl; 0.1% Tween 20).
14. Incubate embryos with 0.5% TNB (TNT + 0.5% blocking buffer, NEN) for 1 h. 15. Replace with 0.5% TNB added with anti-digoxigenin or anti-fluorescein antibody coupled to POD (1:200, Roche). Incubate 1-2 h at RT or overnight at +4° C.
16. Wash 5 times for 20 min in TNT.
17. With mouth pipet, transfer the embryos into a drop (about 30-50 ul) of Amplification Diluent (NEN) for 5 min.
18. Transfer the embryos into a drop of staining solution (tyramid-Cy3 or Cy5 diluted 1/50 in Amplification Diluent, NEN). Incubate in the dark for 50 min, RT.
19. Wash 3-4 times for10 min in TNT.
Embryos can be processed for fluorescent microscopy or stained with the second probe (below).
21. Inactivate the first antibody in 0.1M glycine-HCl pH 2.2, 0.1% Tween 20 for 10 min, RT.
22. Wash 3 times for 5 min in PBT.
23. Incubate with the 2nd antibody coupled to POD (Roche, 1/200), 1-2 h RT or overnight at +4¾C.
24. Repeat steps from #16 and develop with a different colour.
One of the probe reaction can be additionally amplified using the TSA Biotin System (NEN): 25. After step #17, transfer in Biotinyl Tyramid working solution (1/50 of Biotinyl Tyramid stock Solution in Amplification diluent, NEN) for 5-10 min. 26. Wash 3-4 times for 10 min in TNT.
27. Incubate with Streptavidin-HRP (1/100 in TNT) for 30 min at RT or overnight at +4¾C.
28. Repeat from step #16.

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PROBE PREPARATION(for 20 ul reaction)
1 ug of linearized DNA
2 ul Transcription buffer 10X(Roche)
1 mM ATP, GTP and CTP (each)
0.65 mM UTP
0.35 mM Digoxigenin-11-UTP (Roche)
20-50 units of RNase inhibitor
20 units of RNA polymerase

Incubate at 37° C for 2 hours.
Run 2 ul on a gel to quantify RNA (compare to DNA band)
Stop reaction by adding 2 ul of EDTA 0.2 M
Precipitate with 2.5 ul of 4M LiCl and 75 ml of EtOH, 20 min at -70° C.
-Wash with EtOH 70%.
Resuspend in 50 ul of water.
We generally use 5 to 10 ul of probe per ml of hybridization buffer.

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