FISHprotocolsforDrosophila1

.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

• 1x PBS solution.

• PBT solution: 1x PBS, 0.1% Tween-20.

• PBTB solution: 1x PBS, 0.1% Tween-20, 1% milk powder.

4.   Detection of DIG-labeled probe:

a.   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).

b.   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) orAlexa 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