FISH protocols for Drosophila-1
.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 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.
