DOUBLE LABEL IN SITU PROTOCOL(by Kira)
In this example I wanted to stain with flh in black (NBT substrate) and with gsc in orange (INT substrate).
1. Add two differently conjugated RNA probes to the hybridization mix. In this case, I used flh-fluorescein and gsc-dig. Dig-conjugated probes usually work better, so only conjugate fluorescein to your strongest probes.
2. Following hybridization, wash off the probes as usual. Block as usual.
3. Add preadsorbed anti-fluorescein-AP antibody at 1:2000 in 2 mg/ml BSA and incubate for 2 hrs @RT or O/N @ 4_C. Wash off antibody as usual.
4. Equilibrate with NTMT and stain with NBT-BCIP the usual way. In this case, flh staining should come up. Stop the staining reaction with 5 washes in PBT.
5. Wash stained embryos with 0.1M glycine pH 2.2, 0.1% Tween 20 for 30 min @RT to inactivate the alkaline phosphatase. Wash thoroughly with PBT.
6. Block for 1 h @RT in 2 mg/ml BSA, 5% sheep serum.
7. Add preadsorbed anti-digoxigenin-AP antibody at 1:5000 in 2 mg/ml BSA and incubate for 2 hrs @RT or O/N @ 4_C. Wash off antibody as usual.
8. Equilibrate with NTMT. Add 75 _l of INT/BCIP stock solution (Boehringer Mannheim) per 10 ml of NTMT. Stain in the dark until orange-brown stain develops. This usually takes longer than for NBT-BCIP. This time, the gsc staining should come up. Stop the staining with 5 PBT washes.
9. Clear and mount stained embryos in glycerol. Benzyl benzoate/benzyl alcohol will dissolve the INT substrate.
Helpful hints: You should probably try different combinations of conjugated probes, antibodies, and substrates to see which combination works best. A good rule of thumb is to use INT with your stronger probe.
If you are using a really good probe, you could also try using anti-fluorescein-HRP or anti-dig-HRP antibody with that probe, and then you can stain directly with DAB. This will give a stronger brown stain than INT, and DAB is also insoluble in benzyl benzoate/benzyl alcohol.