Post-Hybridization Fluorescent Detection

Detecting Probes with Immunofluorescence:

probe nucleotide label (hapten) or protein (antigen) --- primary antibody --- secondary antibody

So if you have different haptens incorporated into the riboprobes that are now hybridized to your embryos, you can use all of the standard immunofluorescence techniques for double or triple labeling of proteins.

Post-Hybridization Washes and Incubations:

1. Stir the embryos with a finger tap when you arrive in the morning. When it's time to start the post-hybridization washes, warm some hybridization solution to 55° C. When both you and the embryos are ready, remove as much of the probe solution as you can and add 1 ml of the pre-warmed hybridization buffer. Briefly rock the embryos by hand and then put them back to the water bath for 5 minutes.
2. After the embryos have settled, change the hybridization solution (1 ml change) and keep them at 55° C. for 30 minutes. Then change the hybridization solution again and do another 30 minute incubation. During these incubations, periodically take the tube out and rock it briefly by hand until the embryos are stirred up.
3. Rock 1x, 50% PBT / 50% hybridization solution, 10 minutes (now all steps at ROOM TEMPERATURE)
4. Wash 1x, PBT; Rock 3x, PBT, 5 minutes each
5. Wash 1x, (PBT+ Blocking Reagent), 30 minutes

   Now for the fun part, figuring out a set of primary and secondary detection reagents to detect your probes. A full discussion of these possibilities is in the next section below, after the steps of the protocol.

6. Dilute all of the primary detection reagents in (PBT+ Blocking Reagent), using 0.4 ml solution per tube of embryos. Depending on your personal schedule, do one of the following incubations:
   • Rock 2 hours at room temperature OR
   • Rock overnight at 4° C.
   The advantage of doing the longer incubation in the cold is that you can use lower concentrations of primaries, resulting in lower background, and your signals will also be brighter. If you are very impatient like I frequently am, use the shorter incubation and see the embryos today! If you want to detect a protein also, add your primary antibody now along with the others.
7. Wash 1x, PBT; Rock 3x, PBT, 10 minutes each
8. Wash 1x, (PBT+ Blocking Reagent), 30 minutes
9. At this point, depending on your detection scheme, you will either
   • do an enzymatic fluorescent tyramide reaction (see below) OR
   • incubate with fluorescent secondary antibodies (proceed to step 10)
   If you are doing one or more tyramide reactions in addition to using fluorescent secondaries, after the tyramide reaction and washes, come back to step 10.
10. Dilute the secondary antibodies in (PBT+ Blocking Reagent), using 0.4 ml solution per tube of embryos, then Rock 1-2 hours at room temperature. Keep the tubes covered with a little box on the nutator to protect the fluorescent dyes from light during this incubation.
11. Wash 1x, PBT; Rock 4x, PBT, 15 minutes each, still keeping the embryos in the dark, and you are done. Alternatively, do 3 quick PBT washes, and then soak the embryos in PBT overnight at 4° C. Store the tube on its side so the embryos spread to a thin layer, and still protect them from light. In the morning, Rock 1x, PBT, 10 minutes, and you are done.
12. Mount embryos on a microscope slide and enjoy!

Tyramide Signal Amplification (TSA) with Horseradish Peroxidase (HRP):

We follow the instructions that come with the Molecular Probes TSA kits. In brief,

1. Drain the PBT off the embryos
2. Add 100 µl of the fluorescent tyramide solution:
   • 100 µl Amplification Buffer
   • 1 µl Tyramide substrate
   • 1 µl Hydrogen peroxide (100x: dilute the stock according to the kit instructions)
3. Put the tube of embryos in your rack and protect it from light. Let the reaction proceed for 15-20 minutes, periodically stirring up the embryos with a finger tap on the tube.
4. Remove the reaction solution, Wash 3x, PBT; Rock 1x, PBT, 5 minutes
5. At this point, return to the incubation with secondary antibodies at step 10 above. If you're doing two sequential tyramide reactions, see Alternative Methods for Fluorescent Detection.

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Antibody Cocktails and Fluorescent Palettes:

• riboprobes that have different modified nucleotides (haptens) incorporated in them
• primary detection reagents that specifically recognize each hapten
• spectrally distinct fluorescent detection reagents that specifically label each primary detection reagent

After looking at what other people had done with particular reagents to accomplish single and double fluorescent in situs (see Links and References) and a lot of trial and error, this table summarizes what we think make good working combinations that satisfy the three conditions stated above. The dilutions are only recommendations based on what we have tried, so change them according to your own experience. Also, since there are many commercially available anti-hapten antibodies, we have not been able to experiment with them all, and some of them may perform better than those presented here. On the other hand, some other antibodies we have tried without much success and are not presented here, but perhaps they will work in somebody else's hands. If you have another antibody that you know works well to detect a probe, use it. This is a work in progress, but we are satisfied for the moment with this group of reagents that has made possible a quadruple fluorescent in situ. We're using only Alexa-fluor conjugates to do the secondary detection, and not all of the possible fluorochrome/antibody conjugates are listed, only the ones that have been used successfully on embryos; other Alexa secondaries certainly will work. For all these secondaries, I recommend diluting them 1:300-1:500 as a starting point, and then tune your system as you go.

Riboprobe label	Primary detection	Recommended dilution range	Quality	Fluorescent conjugate
Digoxigenin	Mouse anti-DIG	1:300-1:500	++	Alexa 647 Goat anti-Mouse
Digoxigenin	Sheep anti-DIG	1:200-1:400	+	Alexa 488 Donkey anti-Sheep
Digoxigenin	Sheep anti-DIG-POD	1:300-1:500	++	Alexa 568 Tyramide
Biotin	Mouse anti-BIO	1:400-1:800	+++	Alexa 555 Goat anti-Mouse
Biotin	Goat anti-BIO	1:200-1:400	+	Alexa 488 Donkey anti-Goat
Biotin	Streptavidin-HRP	1:200-1:300	++	Alexa 350 Tyramide
Fluorescein	Mouse anti-FITC	1:400-1:800	++	Alexa 488 Goat anti-Mouse
Fluorescein	Goat anti-FITC	1:200-1:400	+	Alexa 488 Donkey anti-Goat
Fluorescein	Rabbit anti-FITC	1:300-1:500	+	Alexa 488 Goat anti-Rabbit
Fluorescein	Rabbit anti-FITC-HRP	1:400-1:600	++	Alexa 350 Tyramide
Dinitrophenyl	Rabbit anti-DNP	1:400-1:800	+++	Alexa 647 Chicken anti-Rabbit

So what should you actually do? You have already chosen your probe labels, so choose the appropriate primaries from different host species, and choose different color fluorescent secondaries that you will be able to excite and image with your microscope. For example, my current favorite combination looks like this:

DIG --- Sheep anti-DIG --- Alexa 488 Donkey anti-Sheep
BIO --- Mouse anti-BIO --- Alexa 555 Goat anti-Mouse
DNP --- Rabbit anti-DNP --- Alexa 647 Chicken anti-Rabbit

I really like using the biotin probes along with Alexa 555, as shown here. The signal is insanely bright and the embryos have essentially zero background. But everything in this table should give you respectable signal and acceptable background if your probes and embryos are good. One word of caution about this particular combination just mentioned: it's possible the Donkey anti-Sheep is recognizing the Goat anti-Mouse to a very slight extent in some experiments, creating a 'shadow' of one image channel in another, similar to the problem of emission bleed-through. If you encounter this problem, it can be fixed by choosing another host species of anti-Mouse. Some other fluorescent detection methods are described in Alternative Methods for Fluorescent Detection.

Addendum (9/03): The cross-reactivity problem mentioned above has now been rectified by choosing a different host species for one of the secondary antibodies. Specifically, the preferred combination of reagents is:

DIG --- Sheep anti-DIG --- Alexa 555 Donkey anti-Sheep
BIO or FITC --- Mouse anti-BIO or Mouse anti-FITC --- Alexa 488 Donkey anti-Mouse
DNP --- Rabbit anti-DNP --- Alexa 647 Chicken anti-Rabbit