Post-Hybridization Fluorescent Detection

Descriptions of all solutions can be found in Reagents and Recipes.

Detecting Probes with Immunofluorescence

This protocol is very similar to alkaline phosphatase-based mRNA in situ staining. After hybridization, essentially the same steps are followed as for protein localization, the difference being that the riboprobe is the target being detected in the following complex:

Probe nucleotide label (hapten) or protein (antigen) --- Primary antibody --- Secondary antibody

Thus, if different haptens are incorporated into the riboprobes being hybridized to the embryos, the standard immunofluorescence techniques for double or triple labeling of proteins can now be employed to visualize multiple mRNA patterns.

Post-Hybridization Washes and Incubations

  1. Following the overnight hybridization, stir the embryos by tapping with a finger. Warm fresh hybridization solution to 55° C., remove as much of the probe solution as possible from the embryos, and then add 1 ml of the pre-warmed hybridization solution. 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. 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.

    At this point, determine which primary and secondary detection reagents are required to detect the probes (see below for a full discussion of the possible choices).

  6. Dilute all of the primary detection reagents in (PBT+ Blocking Reagent), using 0.4 ml solution per tube of embryos. 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 lower concentrations of primaries can be used, resulting in lower background, and signals will typically be brighter. If one wishes to detect expression of a protein also, add the primary antibody along with the others at this point.
  7. Wash 1x, PBT; Rock 3x, PBT, 10 minutes each.
  8. Wash 1x, (PBT+ Blocking Reagent), 30 minutes.
  9. At this point, depending on the detection scheme, one will either:
    • Perform a fluorescent tyramide signal amplification (TSA) reaction (see below) OR
    • Incubate with fluorescent secondary antibodies (proceed to step 10).
    If a TSA reaction is done in addition to using fluorescent secondaries, after the TSA 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. Rock 1-2 hours at room temperature. Protect the tubes from light during this incubation.
  11. Wash 1x, PBT; Rock 4x, PBT, 15 minutes each, still protecting the tubes from light.
  12. Mount the embryos on a microscope slide for viewing.

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

Follow the instructions that come with the Molecular Probes TSA kits. In brief,
  1. Drain the PBT off the embryos.
  2. Add 300 µl of the fluorescent tyramide solution:
    • 300 µl Amplification Buffer
    • 3 µl Tyramide substrate
    • 3 µl Hydrogen peroxide (100x: dilute the stock according to the kit instructions)
  3. Rock the tube of embryos, protecting it from light, and let the reaction proceed for 10-15 minutes.
  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.
Next: Presentation of Embryos for Fluorescence Microscopy

Previous: Hybridization and Making RNA Probes

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Antibody Cocktails and Fluorescent Palettes

Detecting multiple mRNAs separately in one specimen depends on having:

The following table presents the various reagents that have been used to perform single and double fluorescent in situs (see Links and References), as well as what we have added to create the working combinations that satisfy the three conditions stated above. The dilutions indicated are meant only to serve as guidelines, as affinities and concentrations of antibody batches will vary. We list here only some examples of the Alexa-fluor conjugates that have been used successfully in this protocol. The recommended starting dilution for each of the listed fluorescent secondary antibodies is 1:300-1:500.

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 Donkey 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

Once specific labeled probes have been selected, choose appropriate primary antibodies from different host species, as well as spectrally separable fluorescent secondary antibodies and fluorescent tyramides. For example, the following combination works quite well:

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

Also, this combination of fluorescent labels is suitable for imaging on a wide variety of triple-channel confocal microscopes. All combinations shown in this table should give good signals and acceptable background levels if the probes and embryos are in good condition. Some other fluorescent detection methods are described in Alternative Methods for Fluorescent Detection.