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FISH protocols for Drosophila-2

2019.8.20

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 vitro transcription as detailed inStep 2. Care should be taken to work in RNAse free conditions. For mostDrosophila 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), 0.125mL 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.