These are some of the methods we use regularly in the lab and that we thought might prove useful to others in the field. Please feel free to send comments, corrections or improvements. If you're looking for other Drosophila based methods, a good place to start is here.

The procedures described below are from a book chapter PDF in Drosophila: Methods and Protocols, Humana Press, ed: Christian Dahmann, in press. If you wish to cite it, check back later for updated info. Note: microliter designations in the text below are incorrectly shown as 'ml'. Please use the PDF version for proper font usage!

2.1 RNA Probe Preparation (see Note 1)

  1. 1.5 mL microcentrifuge tubes or standard 96-well V-bottom microplates.
  2. RNAse free water.
  3. T7, T3 or SP6 RNA Polymerase (Fermentas Life Sciences, Burlington, ON, Canada; Catalog Nos. EP0101, EP0111, EP0131) as appropriate.
  4. 10x transcription buffer (supplied with polymerases: 0.4 M Tris-HCl, pH 8.0; 60 mM MgCl2, 100 mM dithiothreitol, 20 mM spermidine).
  5. DIG RNA Labeling Mix (Roche Applied Science, Laval, QC, Canada; Catalog No. 11 277 073 910). Recommended for single FISH.
  6. Biotin RNA labeling mix (Roche Applied Science; Catalog No. 11 685 597 910).
  7. RNAguard (Amersham Biosciences, Piscataway, NJ, USA; Catalog No. 27-0816-01).
  8. 3M sodium acetate.
  9. Cold 100% ethanol.
  10. Cold 70% ethanol.

2.2 Embryo Collection and Fixation

  1. Chlorine bleach solution diluted 1:1 with water.
  2. 20 mL glass scintillation vials (Fisher Scientific Limited, Nepean, ON, Canada; Catalog No. 03-337-15) or 1L glass bottle.
  3. 40% formaldehyde solution (prepared on the day of fixing from paraformaldehyde): In scintillation vial, mix 0.92 g paraformaldehyde in 2.5 mL water containing 35 mL of 1N KOH. Dissolve the paraformaldehyde by carefully heating the solution on a stirring hot plate in a fume hood. Once the solution cools down, filter through a 0.45 micron filter and store at 4°C until ready for use. Scale up the recipe if a larger volume is required. (see Note 2).
  4. 1x PBS solution.
  5. Heptane.
  6. Methanol.

2.3 Single FISH on Drosophila embryos

2.3.1 Post-Fixation, Hybridization and Post-Hybridization Washes

  1. 5 mL polypropylene tubes, 1.5 mL and 0.5 mL microcentrifuge tubes, or 0.2 mL half-skirted 96-well PCR plates (Abgene, Rochester, NY, USA; Catalog No. AB-0900).
  2. Microplate sealing foil (Ultident, Saint-Laurent, QC, Canada; Catalog No. 24-PCR-AS-200).
  3. PBT solution: 1x PBS, 0.1% Tween-20.
  4. 40% formaldehyde solution, freshly prepared (Subheading 2.2).
  5. 20 mg/mL proteinase K (Sigma Aldrich, Oakville, ON, Canada; Catalog No. P2308). Dissolve in double distilled water and store aliquots (25-50µL) at -20ºC.
  6. Glycine solution: 2 mg/mL glycine in PBT.
  7. RNA hybridization solution: 50% formamide, 5x SSC, 100 mg/mL heparin, 100 µg/mL sonicated salmon sperm DNA and 0.1% Tween-20. Filter through a 0.2 micron filter and store at -20ºC (stable for several months).
  8. Heating block(s) or water bath(s) adjustable to 56ºC, 80ºC, and 100ºC, or PCR machine.

2.3.2 Development of FISH Signal

  1. 1x PBS solution.
  2. PBT solution: 1x PBS, 0.1% Tween-20.
  3. PBTB solution: 1x PBS, 0.1% Tween-20, 1% milk powder.
  4. Detection of DIG-labeled probe:
    1. Biotinylated anti-DIG antibody followed by Streptavidin-HRP, recommended to obtain strongest signal:Biotin-conjugated mouse monoclonal anti-DIG (1/400 dilution of a 1 mg/ml stock solution in PBTB; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA; Catalog No. 200-062-156) and Streptavidin-HRP conjugate (1/100 dilution of a 1 mg/mL stock solution in PBTB; Molecular Probes, Eugene OR, USA; Catalog No. S991).
    2. HRP-conjugated anti-DIG antibodies, suitable for strongly expressed genes or for double labeling experiments:HRP-conjugated mouse monoclonal anti-DIG (1/400 dilution of a 1 mg/mL stock solution in PBTB; Jackson ImmunoResearch Laboratories Inc.; Catalog No. 200-032-156) or HRP-conjugated sheep monoclonal anti-DIG (1/500 dilution of stock solution in PBTB; Roche Applied Science; Catalog No. 1 207 733).
  5. Tyramide signal amplification:Cy3 tyramide conjugates (1/50 dilution of stock solution in amplification buffer; Perkin Elmer Life Sciences, Boston, MA, USA; Catalog No. SAT704A) or Alexa Fluor 488 tyramide conjugate (1/50 dilution of stock solution in amplification buffer. Molecular Probes; Catalog No. T-20932).See Note 3 for advice on when to use the reagents described in 4 and 5.
  6. 100x DAPI (4’,6-diamidino-2-phenylindole) solution (0.1 mg/mL).

2.3.3 Storage, Mounting and Viewing of Samples

  1. Mountant: 70% glycerol, 2.5% DABCO (1,4-Diazabicyclo [2.2.2.] Octane; Sigma Aldrich; Catalog No. D-2522). In light-shielded tube, add 1.25 g of DABCO crystals, 15 mL of 1x PBS, and 35mL of glycerol and mix on rocking platform until the solution is homogeneous. Store at -20ºC.
  2. Microscope slides.
  3. Coverslips (22x22 mm).
  4. Fluorescence or confocal microscope.

2.4 Double FISH on Drosophila Embryos

  1. Reagents for post-fixation of embryos, probe hybridization, and mounting of samples as described in Subheadings 2.3.1 and 2.3.3.
  2. 1x PBS solution.
  3. PBT solution: 1x PBS, 0.1% Tween-20.
  4. PBTB solution: 1x PBS, 0.1% Tween-20, 1% milk powder.
  5. Quenching solution: 1x PBT, 1% H2O2.
  6. Detection of DIG-labeled probe with HRP-conjugated antibodies:
    • HRP-conjugated mouse monoclonal anti-DIG (1/400 dilution of a 1 mg/mL stock solution in PBTB; Jackson ImmunoResearch Laboratories Inc.; Catalog No. 200-032-156) or HRP-conjugated sheep monoclonal anti-DIG (1/500 dilution of stock solution in PBTB; Roche Applied Science; Catalog No. 1 207 733).
  7. Detection of biotin-labeled probe:
    • Streptavidin-HRP conjugate (1/100 dilution of a 1 mg/mL stock solution in PBTB; Molecular Probes; Catalog No. S991).
  8. Tyramide signal amplification:
    • Cy3 tyramide conjugate (1/50 dilution of stock solution in amplification buffer; Perkin Elmer Life Sciences; Catalog No. SAT704A).
    • Alexa Fluor 488 tyramide conjugate (1/50 dilution of stock solution in amplification buffer. Molecular Probes; Catalog No. T-20932).
See Note 3 for advice on when to use the reagents described in 6-8.

2.5 RNA-Protein Double Labeling

  1. Reagents for post-fixation of embryos, probe hybridization, detection of probes, and mounting of samples as described in Subheadings 2.3.1-2.3.3.
  2. Primary antibody directed against the protein of interest. To prevent antibody cross-detection, make sure that the species origin of this antibody differs from that of the anti-DIG antibody used to detect the FISH probe.
  3. Select a fluorochrome-conjugated secondary antibody directed against the species of the primary antibody.

2.6 FISH on Dissected Tissues

  1. 1.5 mL microcentrifuge tubes.
  2. 1x PBS solution.
  3. 40% formaldehyde solution, freshly prepared (Subheading 2.2).
  4. PBT solution: 1× PBS, 0.1% Tween-20.
  5. Fixation solution: 1× PBS, 4% formaldehyde.
  6. For single or double FISH, prepare reagents for probe hybridization and detection as described in Subheading 2.3.1, 2.3.2, and/or 2.4.

3. Methods

3.1 RNA Probe Preparation

  1. Different strategies can be used to prepare template DNA for synthesizing antisense RNA probes by in vitro transcription.  A gene segment of interest should first be cloned into an appropriate plasmid containing flanking bacteriophage promoter sequences (T3, T7, or Sp6). Then, the plasmid can either be linearized by restriction enzyme digestion or used as a template for PCR to generate an amplified gene fragment with promoter sequences on each extremity. The PCR based approach is particularly useful when templates for several genes are being prepared simultaneously, as most sequences can be amplified using universal primers that overlap the T7, Sp6 and/or T3 sequences. Once the linearized DNA fragments or PCR products have been purified, either through traditional phenol/chloroform extraction combined with ethanol precipitation or agarose gel extraction, they can be used for in vitrotranscription as detailed in Step 2. Care should be taken to work in RNAse free conditions. For most Drosophila genes, cDNA sequences cloned between flanking promoters are readily available in the Drosophila Gene Collections and PCR protocols for the vectors used in these libraries have been described (8). For templates that are amplified in a 96-well plate format, the PCR products can be bulk purified by centrifugation using filter plates (Whatman Inc.; Clifton, NJ, USA; Catalog No. 7700-1303), concentrated by ethanol precipitation and centrifugation in V-bottom 96-well plates, and then resuspended in 15 µL of RNAse-free water.
  2. RNA probes are prepared as described on the product sheets of their DIG and biotin RNA labeling kits (Roche Applied Science). On ice, mix 0.5-1 µg linearized template DNA or PCR product, 2 mL DIG or biotin RNA labeling mix, 2 mL 10x transcription buffer, 1 mL RNAguard (40 U/mL), 2 mL RNA polymerase (20 U/mL), and RNAse-free water to a final volume of 20 mL. Incubate at 37°C for 2-4 h.For PCR templates amplified and purified in 96-well format, probes can be bulk synthesized in V-bottom microplates in a total reaction volume of 10 ml. In each well, 5 mL of resuspended template is combined with 5 mL of pre-mixed and pre-aliquoted transcription reaction mixture containing: 1 mL 10x transcription buffer, 0.5 mL DIG labeling mix, 0.4 mL RNA polymerase (20 U/mL), 0.125 mL RNAguard (40 U/mL), and 3 mL RNAse-free water. Plates are then sealed with adhesive foil and incubated for 2-4 h at 37°C.
  3. Once probe synthesis is completed, RNAse free water is added to the reactions to bring the total volume up to 50 µL, then the probes are precipitated by adding 0.1 volumes 3M sodium acetate and 2.5 volumes of cold 100% ethanol (see Note 4). Place at -70°C over night, then spin and wash the pellets with cold 70% ethanol. After drying, resuspend the probe pellets in 50 mL RNAse-free water. Analyze and quantify the run-off transcripts through conventional agarose gel electrophoresis and ethidium bromide staining. Store probes at -70°C.

3.2 Embryo Collection and Fixation

The following steps can be performed on a small or large scale depending on the size of the fly chambers used for embryo collection.
  1. Prepare 40% formaldehyde stock solution just prior to embryo dechorionation.
  2. Collect and rinse embryos using room temperature tap water and a collection sieve.
  3. Dechorionate the collected embryos in a chlorine bleach solution for approximately 90 s. As dechorionation proceeds, the embryos become clumpy and may tend to stick to the sides of the collection basket. Rinse the embryos immediately and thoroughly with fast flowing room temperature tap water or with embryo rinse solution (0.7% NaCl, 0.03% Triton X-100) to remove residual bleach.
  4. For small collections (<250 µL settled embryos), transfer the embryos to a 20 mL glass scintillation vial containing a biphasic mixture of 8 mL heptane, 2 mL PBS and 200 mL 40% formaldehyde. For large collections (>5 mL of settled embryos), transfer embryos to a 1 L bottle containing 300 mL heptane, 90 mL PBS and 10 mL 40% formaldehyde. Shake for 20 min.
  5. Using a Pasteur or serological pipette, eliminate the lower aqueous phase and most of the upper heptane phase, taking care not to draw up the embryos found at the interface. For small collections, transfer the embryos to a 1.5 mL microfuge tube containing 0.5 mL heptane and 0.5 mL methanol. For large collections, transfer embryos to a 500 mL bottle containing 100 mL heptane and 100 mL methanol. Devitellinize the embryos by shaking vigorously for 45 s until most of the embryos sink to the bottom. Carefully remove most of the heptane and add 1 mL or 100 mL of methanol, for small or large collections respectively. Shake once more. All or most of the embryos should now sink to the bottom of the tube.  Remove all of the liquid along with any unsettled embryos and rinse 3 times with methanol. Embryos can be pooled in polypropylene tubes and stored in methanol at -20°C for several months.

3.3 Single FISH on Drosophila embryos

3.3.1 Post-Fixation, Hybridization and Post-Hybridization Washes

Hybridizations can be performed in 1.5/0.5 mL microfuge tubes (50 mL settled embryos) or 0.2 mL PCR plates (10 mL settled embryos/well). The latter are particularly well suited for optimizing experimental conditions (i.e. antibody titrations) or when many samples are processed in one experiment. Using the recommended PCR plates, which can easily be cut into smaller sections when processing a few dozen samples, greatly facilitates sample manipulation and long term storage. Make sure to seal plates appropriately with sealing foil for all incubations and washes (see Note 5). Unless otherwise indicated, the wash volumes used below are 1 mL or 150 mL for microfuge or PCR tubes, respectively. If not, the appropriate volumes for each tube format are provided, separated by or as above.
  1. Aliquot embryos in tubes or plates (See Note 6).
  2. Rinse the embryos once in methanol, once in a 1:1 mixture of methanol:PBT, and 2 times in PBT.
  3. Post-fix the embryos for 20 min in 4% formaldehyde (prepared by diluting fresh 40% formaldehyde 1/10 in PBT). Place tubes on a rocking platform or rotating mixer to ensure even fixation. If using PCR plates, secure plate in a vertical position to achieve more efficient mixing.
  4. Wash embryos 3 times in PBT for 2 minutes each.
  5. Prepare a working 3 mg/mL proteinase K solution from a 20 mg/mL stock by diluting in PBT. Add 500 mL or 100 mL of proteinase K solution to each embryo sample and incubate at room temperature for 13 min, or adjust the time according to the type of tissue (see Note 7). During this period, mix 5-6 times by gently rotating the tube once or twice or by jetting with a pipetteman. Transfer the embryos to ice and incubate for 1 h. This prolonged incubation on ice ensures uniform penetration and action of the protease.
  6. Remove proteinase K solution and stop the digestion by performing a 2 min wash with a 2 mg/mL glycine solution with rocking. Repeat the glycine wash a second time.
  7. Rinse embryos 2 times in PBT to remove the glycine.
  8. Post-fix the embryos again (as in step 3) for 20 minutes in 4% formaldehyde.
  9. Wash embryos 5 times in PBT for 2 min each to remove all traces of fixative.
  10. Rinse the embryos in a 1:1 mixture of PBT:RNA hybridization solution. Replace the mixture with 100% hybridization solution. At this point, the embryos can be stored for days/weeks at -20°C. If embryos were processed as a large batch (see Note 5), distribute embryos evenly into PCR plates using wide aperture tips, aiming for a final volume of 10 mL settled embryos/well. If 1.5 mL tubes were used up to this point, transfer embryos to 0.5 mL tubes. When ready to hybridize, proceed to step 11.
  11. In a separate tube, boil 400 mL/sample or 100 mL/sample of RNA hybridization solution at 100°C for 5 min, for 0.5 mL or 0.2 mL tubes respectively. Cool on ice for at least 5 min. This freshly boiled hybridization solution will be used as the pre-hybridization solution.
  12. Remove hybridization buffer from embryos. Add cooled pre-hybridization solution and place the embryos in a 56°C heat block/water bath. Incubate at 56°C for a minimum of 2 h.
  13. Prepare probe solution by adding 50-100 ng of probe in 100 mL of hybridization solution, heat at 80°C for 3 min, and cool on ice for at least 5 min. The probe solution can be kept on ice until pre-hybridization is completed.
  14. Remove the pre-hybridization solution and add the probe solution to the embryos. Incubate at 56°C for 12 to 16 h. This step is generally performed overnight.
  15. Pre-heat all wash solutions to 56°C. Remove the probe solution and rinse the embryos once with 400 mL or 100 mL pre-warmed hybridization buffer. Replace the rinse solution with another 400 mL or 100 mL pre-warmed hybridization buffer and incubate at 56°C for 15 min.
  16. Wash for 15 min each with 400 mL or 100 mL of 3:1, 1:1 and 1:3 mixtures of hybridization buffer:PBT.
  17. Wash 4 times for 5 min each, with 400 mL or 100 mL pre-warmed PBT, then cool embryos to room temperature.

3.3.2 Development of FISH Signal

Unless otherwise indicated, the wash volumes used below are 400 µL or 150 mL for 0.5 mL tubes or 0.2 mL PCR strips/plates, respectively. Antibody incubations and washes are performed in PBTB in order to reduce non-specific staining (see Note 8).
  1. Block embryos by incubating with PBTB for 10 min with constant mixing.
  2. Incubate embryos with 300 µL or 100 mL of the appropriate anti-DIG antibody solution for 2 h. (see Notes 3).If an HRP-conjugated antibody is used in Step 2, rinse embryos once with PBTB following the antibody incubation, then perform a nuclear counter stain by incubating for 10 min with a PBTB solution containing 1x DAPI, then proceed directly to Step 6.
  3. Perform 6 washes for 10 min each with PBTB.
  4. Incubate embryos for 1h with 200 mL or 75 mL of streptavidin-HRP solution (diluted 1/100 in PBTB).
  5. Rinse embryos once with PBTB, then perform a nuclear counter stain by incubating for 10 min with a PBTB solution containing 1x DAPI.
  6. Wash the embryos 6 times for 10 min each with PBTB, then once with PBT and 2 times with PBS for 5 min each.
  7. Prepare 1/50 dilutions of the appropriate tyramide conjugate with the amplification buffer supplied in the tyramide kit (see Note 3). Remove the last PBS wash from the embryos, add 150 mL or 50 mL tyramide solution, and incubate in the dark at room temperature for 2 h with constant mixing.
  8. Wash 6 times for 10 min each with PBS.

3.3.3 Storage, Mounting and Viewing of Samples

  1. Resuspend embryos in 200 mL or 100 mL of DABCO mountant. Allow the embryos to settle to the bottom of the tube (1-3 h or overnight at 4°C) before mounting (see Note 9). Embryos can be stored for months/years in microfuge tubes or PCR plates at 4°C in light shielded receptacles.
  2. Transfer a ~35 mL aliquot of embryos, by delicate resuspension using wide aperture tips, onto a clean slide and cover with a 22x22 mm coverslip. Seal the edges with transparent nail polish. Slides can be stored for a few weeks at 4°C in the dark. In our experience, the DAPI stain tends to diffuse away after a few weeks on slides.  Therefore, it is better to mount a fresh aliquot of embryos if samples are re-analyzed at a later date.
  3. Analyze embryos by conventional fluorescence or confocal microscopy.The stainings shown in Fig. 1. exhibit mRNA expression/localization patterns obtained FISH using TSA in comparison with conventional AP-based in situ hybridizations (images from BDGP: http://www.fruitfly.org/cgi-bin/ex/insitu.pl(8).

3.4 Double FISH on Drosophila Embryos

  1. Generate two probes, each with a different label, as described in Subheading 3.1 (see Note 1 for alternative/additional labels).
  2. Collect and fix embryos as described in Subheading 3.2.
  3. Perform the hybridization with both probes simultaneously; all other pre- and post-hybridization washes are as described in Subheading 3.3.1.
  4. Block embryos by incubating with PBTB for 10 min with constant mixing.
  5. Incubate embryos with 300 µL or 100 µL of the appropriate HRP-conjugated anti-DIG antibody solution for 2 h. (see Note 3).
  6. Wash the embryos 6 times for 10 min each with PBTB, then once with PBT and 2 times with PBS for 5 min each.
  7. Prepare a 1/50 dilution of the first tyramide conjugate using the amplification buffer supplied in the tyramide kit (see Note 3). Remove the last PBS wash from the embryos, add 150mL or 50 mL tyramide solution, and incubate in the dark at room temperature for 2 h with constant mixing. All of the following steps should be carried out in a light shielded receptacle.
  8. Wash 6 times for 10 min each with PBS.
  9. Quench the first tyramide reaction by washing for 15 min with quenching solution (see Note 10). Wash 2 times with PBS and 2 times with PBT for 5 min each.
  10. Block embryos with PBTB for 10 min as in Step 4.
  11. Incubate embryos for 1h with 200 mL or 75 mL of streptavidin-HRP solution (diluted 1/100 in PBTB).
  12. Rinse embryos once with PBTB, then perform a nuclear counter stain by incubating for 10 min with a PBTB solution containing 1x DAPI.
  13. Wash the embryos 6 times for 10 min each with PBTB, then once with PBT and 2 times with PBS for 5 min each.
  14. Prepare 1/50 dilutions of the second tyramide conjugate with the amplification buffer supplied in the tyramide kit (see Note 3). Add 150 mL or 50 mL tyramide solution and incubate for 2 h with constant mixing.
  15. Wash 6 times for 10 min each with PBS.
  16. Mount and view samples as described in Subheading 3.3.3.
Fig. 2. shows an example of a double FISH staining for mRNAs encoded by the CG1962 and Canoe genes.

3.5 RNA-Protein Double Labeling

  1. Collect, process, and hybridize embryos essentially as described in Subheading 3.2 and 3.3.1; with the exception of the proteinase K step, which may have to be adapted for optimal immunostaining (see Note 10).
  2. Take care to select non-cross-reactive detection reagents (i.e. antibodies generated in different host species). Add the primary antibody against the protein of interest, along with the appropriate probe detection reagent; HRP- or biotin-conjugated anti-DIG antibodies, or streptavidin-HRP, for DIG- and biotin-labeled probes, respectively. Incubate embros with 300 mLor 100 mL antibody solution (diluted in PBTB) for 2h at room temperature with constant mixing.
  3. Perform 6 washes for 10 min each with PBTB.
  4. Add secondary detection reagents (fluorochrome-conjugated secondary antibodies and streptavidin-HRP). Perform incubations, washes, DAPI staining and TSA reaction as in Subheading 3.3.2. (see Note 11).
  5. Mount samples as described in Subheading 3.3.3.

3.6 FISH on Dissected Tissues

  1. Dissect tissues, such as imaginal disks and salivary glands, in 1× PBS. Dissected tissues can be stored briefly on ice in a 1.5 mL microfuge tube containing PBS until enough tissue is obtained for analysis.
  2. Remove the PBS and add 600 µL of fixation solution. Shake gently for 20 min.
  3. Wash embryos 5 times in PBT for 2 min each to remove all traces of fixative
  4. Perform pre-hybridization, hybridization, antibody incubations, TSA reactions, and mounting of samples as described in Subheading 3.3.1-3.3.3.

4. Notes

  1. The DIG and biotin labels described here can be substituted by or combined with many other labels, including fluorescein, dinitrophenyl and a number of Alexa-conjugated nucleotides. These can be detected by a variety of commercially available antibodies and provide numerous possibilities for multi labeling experiments, as described by Kosman et al.(2004) (7).
  2. Preparing smaller batches of fresh formaldehyde solutions as needed ensures consistent and strong fixation of samples, while avoiding potential loss of activity that might occur with larger volumes of commercially available formaldehyde solutions kept in storage over long periods of time.
  3. To obtain the strongest FISH signal, we recommend using the biotinylated anti-DIG antibody in combination with streptavidin-HRP, which provides an extra signal amplification step compared to the HRP-conjugated anti-DIG antibodies alone. However, though less sensitive, these directly conjugated antibodies are suitable for double FISH experiments, where biotin is used as a second probe label, or for RNA-protein co-detection experiments, where antibody cross-reactivity becomes a concern. While we have mainly used Cy3 and Alexa 488 tyramide conjugates, which both give strong signals; there is a variety of additional fluor conjugated tyramides available from Perkin Elmer Life Sciences and Molecular Probes. The amplification buffer supplied with the Perkin Elmer Life Sciences tyramide kits is in ready to use format. In contrast, when using Alexa tyramide conjugates from Molecular Probes, hydrogen peroxide supplied with the kit needs to be added to the amplification buffer (0.0015% final concentration) before use. The recommended antibody and tyramide dilutions found to be optimal in our laboratory may need to be optimized on a lab-by-lab basis due to variability in research environments and product stocks.
  4. We have found that removal of DNA templates by DNAseI treatment following the transcription reactions, as well as carbonate degradation of probes for increased tissue penetration, to be unnecessary and may risk reducing probe quality. After performing side by side comparisons, we opted for using sodium acetate instead of lithium chloride for probe precipitation, as it provided greater precipitation efficiency.
  5. When intending to use PCR plates for hybridizations, it may be preferable to perform Steps 1-10 in Subheading 3.3.1 using 5 mL polypropylene tubes containing ~300 µl settled embryos (1 tube/quarter plate). This makes the manipulations easier at the proteinase K digestion step where delicate mixing is required and embryos can be aliquoted in PCR plates before starting the prehybridizations. When aliquoting the embryos into PCR plates, it is preferable to use a pipetteman rather then a multichannel pipettor, as it is easier to maintain an even suspension of embryos by up and down pipetting in order to achieve equal embryo distribution in the plates. Take care to eliminate any air bubbles that may have formed under the embryo layer as these may damage the samples during the hybridization step. Once the embryos are aliquoted, multichannel pipettors are recommended for all subsequent washing, antibody incubation and mounting steps. Furthermore, washes can be greatly facilitated by using an 8-well manifold connected to a vacuum pump to aspirate solutions.
  6. When pipetting embryo/tissue samples, wide aperture tips should be used to avoid damaging the embryos. Wide aperture tips can be purchased from a variety of suppliers. If these are not available, simply cut off the ends of traditional tips.
  7. Proteinase K digestion is an important parameter for optimal probe entry into the embryo or tissue of interest. Over digestion can disrupt tissue integrity and morphology, while under digestion can hinder even accessibility of the probe to the entire sample. Traditional protocols suggest a short incubation (1-5 min) at high proteinase K concentration (50 µg/mL); however, we have found that performing the digestions for a longer period of time at lower proteinase K concentrations, followed by a 1h incubation on ice, significantly improves staining sensitivity and uniformity from embryo to embryo. When preparing new proteinase K stocks, or when working with new types of tissue (i.e. dissected tissues, mutant embryos that may be more delicate, etc.), we recommend titrating the concentration of proteinase K in order to find the optimal working concentration. Some tissues, such as dissected larval tissues, tend to be more sensitive to proteinase K digestion; as a result, we often omit the proteinase K step when dealing with such samples. It may also be necessary to reduce proteinase K levels when performing RNA-protein co-staining experiments (Subheading 3.5), as over digestion may perturb epitope recognition.
  8. The concentration of milk used in this protocol has been optimized for use with the antibodies described in Subheading 2.3.2. For other antibodies, it may be preferable to vary the concentration of milk or use alternate blocking reagents (i.e. bovine serum albumin or commercially available blocking solutions) to increase signal specificity.
  9. We find that samples that have been precociously mounted often exhibit a hazy background appearance that dissipates a few hours after the mountant solution has been added to the embryos.
  10. While we have found quenching with 1% hydrogen peroxide to be satisfactory when performing double tyramide reactions, treatment with 0.01M HCl for 10 min or heating at 70°C for 15 min have been suggested as alternative treatments for inactivating the first HRP reaction (6,9).
  11. For RNA-protein co-staining experiments; we have traditionally used secondary antibodies that are directly conjugated to a fluorogenic compound of interest. However, we have begun using TSA as a means of enhancing our immuno-staining signal, through the use of HRP-conjugated secondary antibodies directed against the species of the primary antibody FC fragment, followed by TSA. We suggest testing each approach in parallel to determine which conditions work best on a case by case basis. Most of our secondary antibodies, including both fluor- and HRP-conjugated, were obtained from Jackson ImmunoResearch Laboratories Inc., and are recommended for multi-labeling experiments.
  • Fig. 1. Comparison of alkaline phosphatase and tyramide-amplified detection of mRNAs. Staining patterns obtained using DIG-labeled antisense probes directed against the (A) CG14217, (B,C) Bicaudal-D, and (D) Charybde mRNAs, through conventional AP-based detection (I) or tyramide signal amplification (II), using tyramide-Alexa Fluor 488 (green fluorescence). Close-up images of tyramide-amplified samples are also shown (III). (A) Transcripts of the CG14217 gene demonstrate posterior localization in ring-shaped structures that surround the blastoderm nuclei. (B-C) Bicaudal-D transcripts exhibit highly dynamic localization patterns during Drosophila embryogenesis, including (B) enrichment in perinuclear structures surrounding migrating nuclei and (C) apical localization above peripherally located blastoderm nuclei. (D) During cellularization,Charybde mRNA is detected in nuclear foci representing nascent zygotic transcripts. In rows II and III, nuclei were labeled in red with propidium iodide.
  • Fig. 2. Examples of dissected tissue staining and RNA-protein co-detection. (A) Hybridization performed on Drosophila brain tissue dissected at the pre-pupal stage; using a DIG-labeled antisense probe fordE75 mRNA amplified using Tyramide-Alexa Fluor 488 (green fluorescence. Nuclei were counter-stained in red with propidium iodide. (B-D) Co-detection of fushi tarazu (ftz) mRNA and protein. (B) Embryo hybridized with a DIG-labeled antisense probe for ftz mRNA detected using Tyramide-Alexa Fluor 488 (green fluorescence). (C) Detection of the Ftz protein was accomplished using a polyclonal antibody raised against Ftz and a Cy3-conjugated secondary antibody (red fluorescence). (D) Superimposed images of ftz RNA and protein. Nuclei were labeled in blue with 4'-6-Diamidino-2-phenylindole (DAPI).
This procedure was adapted from one described over the phone (thanks P.G.). We're not sure if it appears elsewhere in published form.

Hoyer's Mountant:

Add 30 g of gum arabic to 50 ml distilled water, stir overnight. While stirring, add 200 g chloral hydrate in small quantities. Add 20 g glycerol. Centrifuge at least 3hr at 12000 g to clear. Add lactate to increase contrast and decrease clearing time. We find 1:4 works best.
  • Wild-type first instar larva cuticle preparation
  1. Collect embryos on apple juice-agar plates for suitable time period from a well-stocked cylinder or cage. Allow to age for 24-36 hr at 25ºC.
  2. Rinse unhatched larvae into a nytex screen. Dechorionate in 3% bleach until embryos float to the surface (1-5 min). Don't worry about over-dechorionating. Rinse with water.
  3. Remove screen and dip into a scintillation vial containing 5ml PBS/5ml heptane. The embryos should slide off. Those with vitelline membranes intact will stay at the interphase, while hatched larvae will settle to the bottom. Use a paint brush to remove remaining larvae from the dechorionating vessel and dip into heptane layer to dislodge from brush.
  4. Using a 1ml pipetteman, suck up vitelline containing larvae from interphase. Eject any of the lower aqueous phase sucked up in the process. Transfer embryos in upper heptane solution to an eppendorf tube. Adjust volume to approximately 0.5 ml and add an equal volume of methanol.
  5. Close cap and shake vigorously for 15 seconds to devitellenize. The majority of embryos will settle to the bottom. Remove most of upper phase without removing any embryos. Shake once again. Now all of the embryos should settle to the bottom. Remove liquid and wash 2-3x with methanol.
  6. Larvae at the bottom of the scintillation vial are generally wild type. They can also be drawn up in the aqueous solution in a P-1000 and transferred to an eppendorf tube. Remove the majority of liquid and add around 0.5 ml of methanol. Larvae should settle. Rinse 2X more with methanol. Larvae can be pooled with those above if desired.
  7. Again using a P-1000, transfer the larvae to a clean glass slide. Use the excess methanol to disperse the larvae evenly by adding drops over clustered larvae. Allow the methanol to air dry briefly and then add a drop of Hoyer's/lactate. Cover carefully with a suitably sized coverslip, taking care to avoid bubbles.
  8. Place in a 65ºC oven overnight to clear the larvae. Once cleared, flatten the larvae as follows. Wrap in a layer of aluminum foil. Place on a flat surface, cover with a second glass slide and place a lead pig such as those which come with radioactive substances. With this weight, larvae should be suitably flattened within 1-4 hr. Take care not to move the coverslip while adding or removing the weight and foil, as this will destroy the cuticles. Upon removing the foil, excess Hoyer's should have been exuded from the slide. This can be cleaned away using first water and then ethanol squirted in a stream over the slide. This avoids physical contact which may move the coverslip. Allow the slide to air dry and seal the edges with your favorite nail polish. Once dry, the slide should be permanent and relatively resistant to physical abuse.
This protocol is described in less detail in Copeland et al, Nature, 379, 162-165, 1996.
Affinity chromatography can be performed using a number of different protein tags. In our lab we have had success using poly-hisitidine tagged proteins for creation of our protein affinity columns. The histidine tag is very short (10 his residues) and should not alter the conformation of the tagged protein, nor should it be involved in artifactual interactions. The poly-his tag binds to a nickel chelate resin for creation of the column. We use the resin alone for the control columns. Most often, we challenge our columns with 35S-labeled, in vitro translated proteins, however, proteins from a variety of sources can be used and detected appropriately. The protocol below describes our method. The order of the steps has been optimised so that both the affinity columns and in vitro translated protein are ready at the same time.
  • Affinity chromatography with FTZ (1st 3 lanes) and control (2nd 3 lanes) affinity columns. 35S-labeled PRD and FTZ-F1 bind to the FTZ columns and can then be eluted (E). (L=load, FT=flowthrough; Luc=luciferase, negative control).
  1. Begin by getting out the DNA and 35S-methionine for the in vitro translation reactions.
  2. While these thaw, prepare solutions for affinity columns:
    • AC Buffer:(10% glycerol, 100mM NaCl, 20mM Tris pH 7.6, 0.5mM EDTA, 0.1% Tween-20, ddH2O).NOTE: You may wish to add protease inhibitors (eg. TPCK), but usually we don't; you may also choose to filter sterilize the buffer, but again, usually we don't.
    • 5% milk powder solution: 10 mL AC Buffer + 0.5g milk powder. Mix on a shaker platform until dissolved.
    Store both solutions on ice.
  3. Prepare the nickel resin. 50μL (settled volume) of resin is required for each experimental and control column. Transfer the appropriate volume of slurry into two siliconized eppendorf tubes, one for the control resin, one for the protein coupling. The resin should be allowed to settle and then washed once in water and twice in AC buffer. In each wash step the resin can be allowed to settle by gravity or by centrifuging at 1.5K for 3 minutes in a benchtop centrifuge.
  4. Between washes of the resin begin to set up the in vitro translation reactions for the proteins that are to be tested for binding. We follow the standard protocol for a 25μL reaction using the Promega TNT coupled transcription/translation kit.
  5. While the reactions are incubating couple your protein to the resin. We use 1mg of pure His-tagged protein per mL of resin. However, the optimal ratio may vary depending on the interaction being pursued. We treat the control resin with the same buffer that the protein is stored in. Allow the coupling to proceed for 30 minutes on ice.
  6. Prepare spin columns for removal of unicorporated 35S-methionine from the translation reaction. The columns are made in 1 mL syringes blocked with glass wool. The resin is G-25 (BioRad) equilibrated in AC buffer. Follow the Maniatis protocol for spin columns.NOTE: in the following steps, you want to avoid foaming and bubbles as much as possible, as the bubbles can contribute to protein denaturation. So, always mix gently.
  7. While the protein is being coupled, and between spins in the preparation of spin columns, begin to set up the housings for the affinity columns. We use 200μL tips with bevelled ends (Diamed #230-1673-03K). The tips have to be blocked with glass beads to prevent the resin from leaking out. Use Sigma Acid washed 212-300 micron glass beads that have been washed in water. Beads should be added to a level of 7mm at the end of the tip. After the beads have been transferred, remove excess water and then add 100μL of AC buffer. This is to prevent the contact of protein resin directly with water.At this time the fraction collection tubes can also be labeled. The columns are run standing in eppendorf tubes on ice, the columns are moved to new tubes for each fraction. 7 collection tubes will be needed for each column: 1 flow-through fraction, 4 wash fractions, and 2 elution fractions.
  8. When the in vitro translation reactions are finished pass the reaction over the spin column in a volume of 100μL.
  9. Set up the column load sample. 50μL in vitro reaction (half the sample after the spin column), 130 μL AC buffer, and 45μL 5% milk powder. Total volume=225 μL
  10. Wash the nickel resin. After the protein has been allowed to couple to the resin for 30 minutes on ice, wash the resin twice in 1mL of AC buffer to remove unbound protein and to equilibrate the resin in AC buffer.
  11. Set up the columns. Add 50μL of settled affinity resin to the prepared tips. Allow the resin to settle on ice.
  12. Load the columns. After the resin has settled (5-10 minutes) remove any buffer remaining on top. Carefully load 90μL of the load sample to the top of each column (control and experimental). DO NOT DISTURB THE SETTLED RESIN. The remaining portion of the load sample should be saved for SDS-PAGE.
  13. Allow the loaded protein to flow into the resin. When it reaches the bottom of the column (you can follow its progress because of the red color of the retic. lysate) transfer the column into tube #1. Allow the remaining protein to flow into the tube. This comprises the majority of the flow-through fraction. When the last of the load has flowed into the column transfer the column to tube #2.
  14. Wash the columns with 4x400μL of AC buffer. Each wash fraction is 400μL. Transfer the column to a new tube after each wash step.
  15. Elute the bound protein. After the last wash transfer the columns to tube #6 at room temperature. Add 30μl of 2% SDS (this would precipitate on ice). Allow this to flow-in. Transfer the column to tube #7. Add 120μl of 2% SDS. Allow this to run through. This is the eluate fraction.
  16. For gels: Load and Flow Through will have 9 μL sample + 3 μl 4X SDS gel loading bufferTo compensate for dilution, eluate loaded will equal 15 μl sample + 5 μL 4X SDS
  17. Boil the samples, store in freezer until ready for SDS-PAGE. After electrophoresis, dry the gel and autoradiograph. An interaction is detected as depletion of the labeled protein from the flow-through and presence of the protein in the experimental column eluate.
This protocol is used in two recent publications (Guichet et al,1997; Schwartz et al, 2001).
  1. Run samples out on a gel. For bacterially expressed proteins, generally 5 uL is plenty (1ml cell culture; cells resuspeded in 50 μl loading buffer). Run the gels (BioRad mini gels) at 195 V for approximately 40 min. (until samples run close to the bottom of the gel).
  2. Transfer the proteins from the gel onto nitrocellulose. For the BioRad setup, the case should be set up as follows: black side down, then 3M Scotch Brite Pad, then blotting paper, gel, nitrocellulose, 2nd blotting paper, 2nd Scotch Brite Pad, and the clear side of the case. Put the case in the holder, black side of the case facing black side of the holder. Run the transfer at 100V for 1 hour.
  3. Meanwhile, prepare 500 mL of AC Buffer (+ Tween):
    • 50 mL glycerol (= 10% glycerol)
    • 10 mL 5M NaCl (= 100mM NaCl)
    • 10 mL 1M Tris, pH 7.6 (= 20 mM Tris)
    • 1 mL 0.5M EDTA (= 0.5mM EDTA)
    • 5 mL 10% Tween-20 (= 0.1% Tween-20)
    • put on ice
    Make 50 mL (or more) of 2% milk powder solution:
    • 50 mL AC Buffer
    • 1 g milk powder
    • put on a rocker to dissolve the milk powder (may take 20-30 minutes). Then put on ice.
  4. Make the probe, using the TnT (Promega) Reticulocyte Lysate kit. Set up either a 25 uL reaction or a 50 uL reaction, depending on the size of tray you'll be using for washes and probing. Below is the recipe for a 50 uL reaction mix:
    • 25 uL Reticulocyte lysate (I use a little more, ~27 uL)
    • 2 uL Reaction Buffer
    • 1 uL T7 (or T3) polymerase (or other polymerase)
    • 1 uL amino acids minus methionine (or missing other amino acid)
    • 1 uL RNA Guard
    • 4 uL 35S-met (or other labelled amino acid)
    • 16 uL DNA + ddH2O (1-2 ug DNA)
    • Spin down the sample (pulse spin) to remove air bubbles. Let the reaction proceed at room temperature for 1 hour, 10 minutes (can be longer or shorter, depending on the protein).

Block, Probe, and Wash

  1. After the transfer is complete, put the blot into a tray. Keep the side that was touching the gel up). Do 1-2 quick washes with 1X PBS to remove the SDS. Then, do a quick rinse in AC Buffer, to remove the PBS. Pour on the 2% milk powder (just enough to cover the blot), put a lid on the dish, and rock the blot at 4 C for 1 hour. This is the blocking step.
  2. Meanwhile, set up 2 spin columns:
    • Use 1cc syringes, remove the cap and the plunger. Put in glass wool to fill the opening at the bottom, push down with the plunger. Put the syringe in a 15 mL falcon tube. Add BioRad G 25 resin, stored at 4 C in TE buffer, to the syringe , filling to the top (avoid air bubbles!). Spin down at 2000 rpm for 3 minutes. Then refill with G-25, and repeat the spin cycle. (At this point, the G-25 should be packed down such that it occupies about 0.8ml.
    • Add to the columns 100 uL AC Buffer, spin at 2000 rpm for 3 min. Repeat two more times.
  3. When the probe labeling reaction is finished, dilute the sample using AC Buffer to make a final volume of 100 uL. Load this on the spin column and spin at 2000 rpm for 3 minutes in a fresh falcon tube. Collect the flow-through. This process removes unincorporated nucleotides.
  4. When there are approximately 20 minutes left in the blocking step, it is time to prepare the probe mix. At this point, you may want to collect a 2uL sample of your probe (from the 100 ul), and combine it with 10 uL 1X SDS gel loading buffer. You can then run this sample on a gel, and put film on the dried gel to see if the probe labelling step actually worked. The volume of the probe mix depends on your initial probe reaction mix volume, and the size of tray that you are using. For example, if you set up a 50 uL initial reaction, you can use up to 20 mL of probe mix. For a 25 uL reaction, use no more than 10 ml of probe mix.
Small Tray Large Tray
10 mL 2% milk powder solution 20 mL 2% milk powder solution
10 uL 1M DTT 20 uL 1M DTT
25 uL probe reaction 50 uL probe reaction
Let the probe incubate in the mix for the remaining 20 minutes, on ice.
  1. When the blocking step is over, pour off the milk powder solution, and pour on the probe mix. Cover the tray. Rock the blot in the cold room for 2-3 hours.
  2. When you are finished probing, pour the probe mix into a suitable radioactive waste container. Then do 2 quick washes with AC Buffer. Wash with remaining 2% milk powder solution (+ DTT), and let the blot rock in the cold room for 15 min. (make sure you have enough solution to cover the blot completely).
  3. Do a quick wash in AC Buffer, and follow with 4 x 20 min washes in AC buffer, in the cold room. (use at least enough to cover the blot)
  4. Let the blot dry on blotting paper. After 30-40 minutes, transfer to a new piece of blotting paper. Add baby powder to prevent sticking. Spread GENTLY. Make sure the side of the membrane that was in contact with the gel is the side that is up. Put on x-ray film, and expose.

Notes:

  • if preferred, the blocking step can go overnight (the step before probing), rather than for an hour. So, if you are pressed for time, this is one point where you can spread the Far Western over two days.
  • Denaturation/Renaturation steps can also be tried to optomize signal: noise. For example, right after the transfer, the blot is washed with 6M Guanidine-HCl in AC Buffer plus 2% milk powder (and DTT) for 30 minutes at room temperature (use 8M stock). Then it is washed in 3M Guanidine-HCl in 2% milk powder (in AC Buffer) for 30 minutes at RT; then 1M Guanidine-HCl in 2% milk powder (in AC Buffer) for 30 minutes at 4 C, and then in 0.1M for 30 minutes at 4 C. Finally, it is put in 2% milk powder solution, in AC Buffer, with DTT, overnight at 4 C. Proceed with probing the next morning.
  • Below is a table showing how to make the Guanidine-HCl solutions.
[guanidine-HCl]
Reagent 6M 3M 1M 0.1M 2% M.P.
Glycerol 2.5 mL 2.5 mL 2.5 mL 2.5 mL 2.5 mL
5M NaCl 0.5 mL 0.5 mL 0.5 mL 0.5 mL 0.5 mL
1M Tris, PH 7.5 0.5 mL 0.5 mL 0.5 mL 0.5 mL 0.5 mL
0.5M EDTA 0.05 mL 0.05 mL 0.05 mL 0.05 mL 0.05 mL
10% Tween-20 0.25 mL 0.25 mL 0.25 mL 0.25 mL 0.25 mL
8M Guanidine-HCl 18.75 mL 9.30 mL 3.13 mL 0.31 mL -
Milk Powder 0.5 g 0.5 g 0.5 g 0.5 g 0.5 g
1M DTT 25 μL 25 μL 25 μL 25 μL 25 μL
ddH2O 2.45 mL 12.82 mL 18.07 mL 20.89 mL 21.20 mL
Total Volume 25 mL 25 mL 25 mL 25 mL 25 mL
  • Far Western blot of FTZ polypeptides expressed in bacteria and detected by 35S FTZ-F1.
  1. Collect embryos, dechorionate and fix in a (1:1) mixture of heptane:fix. Fix is 5% formaldehyde, 0.05M EGTA in 1X PBS. pH 7.0. Devitellinize embryos by shaking in an equal mixture of methanol:heptane (embryos will sink to the bottom) and wash the embryos well with methanol. Transfer the embryos to a 0.5 ml eppendorf tube.
  2. Wash the embryos with 3% hydrogen peroxide in methanol for 15 min. Rinse well in methanol.
  3. Wash 3 X 30 minutes with PBTH (filtered 1 X PBS, 0.1% Tween-20, 50 ug/ml heparin, and 250 ug/ml tRNA). You can also include 1% BSA providing you know it is ultra pure/RNase-free. With our reagents, we find we get similar results without it.
  4. Incubate with the primary antibody (diluted appropriately in PBTH) overnight at 4C. For extra RNase protection, include 2- 5 Units of RNasin/250 ul of antibody mixture.
  5. Wash at room temperature with several changes of PBTH for at least 1-2 hr.
  6. Incubate with biotinylated secondary antibody (diluted in PBTH) for 1.5 hours at room temperature (again add RNase inhibitor).
  7. Wash with several changes of PBTH for 60 minutes. For detection of the biotinylated secondary antibody, we generally use the Vector Lab ABC amplification system. Mix the reagents per manufacturer's instructions 30 min. prior to use.
  8. Incubate embryos with the pre-incubated ABC mixture for 30 minutes at room temperature.
  9. Wash for 15-30 min. with 3-4 changes of PBTH. Develop the signal with 20 ug/ml Diaminobenzidine, 0.03% hydrogen peroxide in PBTH. This will produce a brown signal. Stop the reaction with several rinses of PBTH.

In situ Hybridization:

  1. Fix the embryos for 20 minutes with 5% formaldehyde in PBT (PBT= filtered 1 X PBS with 0.1% Tween-20) at room temperature.
  2. Wash 3 X 5 minutes with PBT.
  3. Incubate the embryos with 50 ug/ml of Proteinase K in PBT for 2-3 minutes (time varies with different Prot. K preps and needs to be optimized).
  4. Stop the Proteinase K reaction with two washes of 2 mg/ml glycine in PBT. Each wash should be 2 -3 minutes. Then rinse the embryos twice with PBT.
  5. Fix the embryos again for 20 minutes with 5% formaldehyde in PBT at room temperature.
  6. Wash the embryos 5 times with PBT.
  7. Rinse the embryos with PBT:Hybridization buffer (1:1), and then with hybridization buffer alone. Hybridization buffer for DNA probes is: 5 X SSC, 100 ug/ml sonicated, boiled salmon sperm DNA, 100 ug/ml tRNA, 50 ug/ml heparin, and 0.1% Tween-20 (for RNA probes and hybridization conditions, see for ex. our protocol on FISH whole-mount staining).
  8. Pre-hybridize the embryos in hybridization buffer for at least one hour at the appropriate temperature (48 C for DNA probes). Longer pre-hybridizations of up to 3 hours seem to improve the signal to noise ratio.
  9. Add your Digoxygenin-labeled probe to the hybridization buffer and incubate overnight (12 to 16 hours) at the appropriate temperature. Use about 100 ul hybridization buffer per 50 ul settled embryos.
  10. Carry out the following post-hybridization washes at the hybridization temperature for at least 20 minutes each:
    1. hybridization buffer alone
    2. PBT:hybridization buffer (1:1)
    3. PBT alone for at least 5 washes
  11. Incubate with anti-Digoxygenin antibody (Boerhinger Mannheim) conjugated to alkaline phosphatase (diluted 1:2000 in PBT) for 2 hours at room temperature.
  12. Wash with PBT 4 times for 20 minutes each wash. Rinse with alkaline phosphatase staining buffer (100mM NaCl, 50 mM MgCl2, 100 mM Tris pH 9.5, 1 mM Levamisol, and 0.1% Tween 20).
  13. Develop the colour reaction using NBT and BCIP in the alkaline phosphatase staining buffer as described by the manufacturer (Boehringer Mannheim).
  14. Stop the reaction with several washes of PBT and dehydrate with an ethanol series (in PBT).
  15. Rehydrate the embryos in a reverse ethanol series (in PBT), and then rinse several times with PBT.
  16. Mount in 70% glycerol and 30% 0.1 M Tris pH 8.0.