Krause Lab Protocols
Single and Double FISH protocols for Drosophila
This procedure is taken from a recently published book chapter. The reference is: Hughes, S. and Krause, H.M. (1998) Single and double FISH protocols for Drosophila. in: Protocols in confocal microscopy., ed. Stephen Paddock, Humana Press Inc., in press.
1.1 RNA Probe Preparation
1. 1.5 ml microcentrifuge tubes, autoclaved.
2. RNAse free DEPC treated or double-distilled water.
3. 5X T7/T3 transcription optimized buffer (Promega, Madison WI, USA; Cat No. P1181)
4. T7 or T3 RNA Polymerase (Promega, Madison WI, USA: Cat Nos. P2075, P2083; 1000 U).
5. Fluorescein RNA labeling Mix (Boehringer Mannheim; Cat No. 1685 619).
6. Digoxigenin RNA labeling Mix (Boehringer Mannheim; Cat No. 1277 073).
7. RNAguard (Pharmacia; Cat No 27-0815-01)
8. 0.5 M EDTA.
9. 4M lithium chloride.
10. Absolute ethanol.
11. Cold 70% ethanol wash
1.2 Initial Embryo Fixation
1. Chlorine bleach; diluted 1:1 with water.
2. 40% formaldehyde solution (prepared fresh from paraformaldehyde as described below).
3. 10X PBS solution.
6. 20 ml Disposable Glass Scintillation Vials (Fisher).
7. 1.5 ml microcentrifuge tubes, autoclaved.
1.3 Post- Fixation and Hybridization of Whole-mount Embryos
1. PBT solution: 1X PBS plus 0.1% Tween-20.
2. 40% formaldehyde solution, prepared that day).
3. 20mg/ml Proteinase K (Sigma); dissolve in sterile H2O, divide into 50 ul aliquots and store at -20oC) .
4. 2mg/ml glycine in PBS.
5. RNA hybridization solution: 50% formamide, 5X SSC, 100 ug/ml heparin, 100 ug/ml sonicated salmon sperm DNA and 0.1% Tween 20). Filter through a 20 micron filter and store at -20oC in aliquots (stable for at least 6-12 months).
6. Hot block or water bath at 80oC.
7. Water bath at 56oC.
1.4 Post-Hybridization Washes and Development of the FISH signal
1. RNA hybridization buffer.
2. PBT solution: 1XPBS, 0.1% Tween-20.
3. PBTB solution: 1XPBS, 0.1% Tween-20 and 0.5% milk powder.
4. Mouse monoclonal anti-fluorescein antibody (IgG: 1/2000 dilution of a 0.1 mg/ml stock solution [see notes 2,3]; Boehringer Mannheim, Laval, QC, Canada; Cat No. 1426 320).
5. Sheep anti-digoxigenin antibody (IgG; 1/1000 dilution of a 0.2 mg/ml stock solution; Boehringer Mannheim, Laval, QC, Canada; Cat No. 1333 089).
6. Goat anti-mouse antibody conjugated to CY2 [F(ab')2 fragment of IgG (H+L) 1/2000 dilution of a 1 mg/ml stock solution; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA; Cat No. 115-226-062].
7. Donkey anti-sheep antibody conjugated to CY3 [F(ab')2 fragment of IgG (H+L); 1/2000 dilution of a 1mg/ml stock solution; Jackson ImmunoResearch Laboratories Inc., West Grove. PA, USA.; Cat No. 713-166-147].
8. Embryo mountant: 70% glycerol, 2% DABCO (1,4-Diazabicyclo [2.2.2.] Octane; Sigma, USA; Cat No. D-2522).
9. Microscope slides.
10. Coverslips (22 X 50 mm).
11. Fluorescence and/or LSC microscope.
2.1 RNA Probe Preparation
1. To prepare run-off transcripts, the plasmid template is first linearized to completion (see Note #1) with the appropriate restriction enzyme, and then the enzyme removed by careful phenol and then choloroform extractions. After removal of all chloroform (heating to 65oC for 15 min helps), precipitate the DNA by adding NaAcetate (pH 5.2) to 0.3M, three volumes of ethanol, and cooling to -70oC for 20 min. Spin 10 min in a cold microcentrifuge and wash with cold 70% ethanol. We generally prepare 5-10 mg linearized template, resuspended in 20 ul RNAse-free water.
2. RNA probes are prepared as described by Boehringer Mannheim on their digoxygenin and fluorescein RNA labeling spec sheets. On ice add 1 ug of linearized template DNA (3-5kb), 2ul fluorescein or digoxigenin RNA labeling mix, 2 ul 10X transcription buffer (supplied with the RNA polymerase: 0.4M Tris-HCl, pH 8.0; 60 mM MgCl2, 100 mM dithiothreitol, 20 mM spermidine), 1ul RNAse inhibitor (1unit/ul) and sterile, RNAse-free water to make the final reaction volume equal to 18 ul. Add 2 ul of the appropriate RNA polymerase (T7 or T3), mix well and incubate at 37oC for 2 hours.
3. Following the transcription reaction (see Note #2), the labeled probe is precipitated by addition of 1 ul of 0.5M EDTA, 2.5 ul 4M LiCl, and 75 ul absolute ethanol. Chill to -70oC, spin and wash the pellet as described above. After drying, resuspend the pellet in 100 ul RNAse-free water (see Note #3). Check the probe by loading and running 4-5ul on a conventional agarose gel (~1%). The run-off transcript should easily be detected by ethidium bromide staining (see for ex. Hughes and Krause, 1998). Probe should be stored at -20oC. Several freeze/thaw cycles on ice do not impair probe activity.
2.2 Initial Embryo Fixation
1. Prepare 40% formaldehyde stock solution just prior to embryo dechorionation (see Note #4). Disolve 0.92 gm paraformaldehyde in 2.5 ml of water containing 35 ul 1N KOH. Heat the mixture at 80oC until dissolved.
2. Collect and rinse the embryos in water.
3. Dechorionate the collected embryos in a 1:1 mixture of chlorine bleach and water for approximately 90 seconds. When dechorionated the embryos will either float to the surface of the bleach solution or stick to the sides of the collection basket. Embryos should be rinsed immediately as over-dechorionation is apparently detrimental. Rinse the collection basket with plenty of water. Fast flowing tap water can help dechorionate partially dechorionated embryos. An optional rinse with 0.7% NaCl, 0.03% Triton X-100 is helpful for removing residual bleach and for washing embryos down from the side of the basket.
4. Transfer the embryos to a 20 ml glass scintillation vial (see Note #5) containing a two phase mixture of 8 ml heptane, 2.5 ml 1XPBS and 250ul 40% formaldehyde. Shake for 20 minutes.
5. Using a 1ml pipetteman, draw up embryos (which are at the interphase), taking care not to suck up any of the lower aqueous phase (see Note #6 ). Transfer to a 1.5 ml microfuge tube containing 0.5 ml heptane and 0.5 ml methanol for devitilinization. Shake vigorously until the majority of the embryos sink the bottom (about 30 seconds). Carefully remove about 75% of the heptane and methanol and replace with 1ml methanol. Shake once more. All or most embryos should have now sunk to the bottom of the tube. Remove all liquid along with any embryos at the interphase, if present, and then rinse 2-3 times with methanol. Embryos can be stored in methanol at -20oC for several months.
2.3 Post-Fixation and Hybridisation of Whole-mount Embryos
The following steps are optimized for ~ 50 ul settled embryos in a 1.5 ml microfuge tube.
1. Rinse the embryos once in methanol.
2. Rinse the embryos twice in PBT (1XPBS, 0.1% Tween 20).
3. Post-fix the embryos for 20 minutes in 0.5ml PBT containing 50 ul freshly prepared 40% formaldehyde. Place tubes on a rocking platform to ensure even fixation.
4. Rinse embryos three times in PBT. Washes should be approximately two minutes in duration.
5. Add approximately 0.5ml PBT containing 50 ug/ml non-digested Proteinase K. Incubate for ~1 to 1.5 minutes (see Note #7). Mix by drawing up some of the solution with a pipetteman and gently jetting the embryos back into suspension. Repeat once, allow embryos to settle and then remove the solution at least 30 seconds before the end of the incubation period.
6. Stop the Proteinase K digestion by immediately adding 1ml of PBT containing 2mg/ml glycine. After about 2 min, remove and rinse for another 2 min in the same solution.
7. Rinse embryos twice in PBT to remove the glycine.
8. Post-fix the embryos once again (as in step 3) for 20 minutes in PBT containing 4% formaldehyde.
9. Wash the embryos extensively in PBT to remove all traces of fixative.
10. Rinse the embryos in 1ml of 50% PBT; 50% RNA hybridization solution. Replace the mixture with 100% hybridization solution and pre-hybridize the embryos at 56oC for a minimum of 2 hours.
11. After pre-hybridization, place the embryos in a sterilized 0.5 ml microfuge tube, remove prehybridization solution and add probe. Optimal probe concentration needs to be determined empirically but generally 1 ul probe in 200 ul RNA hybridization solution works well. Diluted probe is heated to 80oC for 3 minutes, cooled briefly on ice and then added to the embryos.
12. Hybridizations are carried out at 56oC for 12 to 16 hours. Mix embryos 2-3 times during the course of the incubation, either by quickly inverting the tube, or by using a pipetteman to gently jet the embryos into suspension.
2.4 Post-Hybridisation Washes and Development of the FISH signal
1. Remove any Hyb solution and embryos from the upper walls and cap of the microfuge tube by spinning the tube for ~ 10 seconds at 1,500rpm in a microcentriguge.
2. Remove the probe solution and rinse the embryos once with 400 ul pre-warmed hybridization buffer. Add another 400 ul pre-warmed hybridization buffer and incubate at 56oC for 20-30 minutes. Invert tube several times.
3. Wash for another 20-30 min with a 1:1 mix of hybridization buffer and PBT and then with four 5 min washes of PBT. All washes should be done with pre-heated solutions at 56oC .
4. Cool to room temperature and incubate for 10 min in 400 ml PBTB (1X PBS, 0.1% Tween 20, 0.5% milk powder). The use of milk powder in this and subsequent steps helps reduce background.
5. Hybridized RNA probes are detected by first incubating the embryos with the appropriate primary antibodies diluted in PBTB. For double-labeling, both anti-digoxygenin and anti-fluorescein antibodies are added. Dilutions (see Note #8) that were optimal in our hands are given in the materials section above, but batches may vary, as may optimal activity given the many variations that exist in a particular lab's reagents, equipment and methodology. Incubate with primary antibodies for 2 hr (optionally overnight at 4oC), with constant mixing on a rocking platform or rotating mixer.
6. Wash for 1-2 hr with 4-5 changes of PBTB.
7. Add the appropriate (see Note#9) secondary antibody(s) diluted in PBTB, and incubate with constant mixing for 2 hr. Carry out this step and all subsequent steps in dim light with tubes covered or wrapped in foil.
8. Wash for 2 hr with 4-5 changes of PBTB and then finally with PBT.
9. Resuspend embryos in DABCO-containing mountant. Allow the embryos to settle to the bottom of the tube (1-3hr or overnight at 4oC) before resuspending and mounting.
10. Transfer the embryos to a clean slide in ~ 80 ul mountant and cover with a 25 X 50 mm coverslip. Seal the edges with nail polish. Slides can be stored for weeks at 4oC in the dark. Background levels will often decrease over the first few days.
11. Embryos can be viewed by either conventional fluorescence microscopy, LSCM or deconvolution microscopy. Basic LSCM techniques are discussed elsewhere in this book.
2.5 RNA-Protein Double-labeling
1. Collect and fix embryos as described above for FISH, with the exception of the Proteinase-K step. The Proteinase-K concentration may have to be lowered to preserve integrity of protein epitopes (see Note # 7).
2. After performing the hybridization and washes, as described, add the primary antibody for the protein of interest, along with the anti-fluorescein or anti-digoxygenin antibody. To obtain differential signals, the protein antibody must have been raised in a host other than the host(s) used for the probe-specific antibodies (ie, not mouse or sheep).
3. After primary antibody incubation and washes, the primary antibodies are detected using appropriate secondary antibodies (see Note #9). Wash and mount as with single or double FISH staining. With careful choice of antibodies, triple staining a combination of transcript and protein targets is possible. However, secondary antibodies conjugated to CY5 are necesary (ref), as are the microscope excitation and detection components required for visualization.
2.6 Performing FISH on Dissected Tissues
1. Dissect tissues such as imaginal disks or salivary glands in PBS. Dissected tissues can be stored briefly (up to 30 min) on ice in a microfuge tube containing PBS while collecting enough tissues for analysis.
2. Remove the PBS and add 50 ul 10X PBS, 325 ul water, 500 ul heptane and 125 ul 40% formaldehyde (freshly prepared as described above). Shake gently for 45 seconds.
3. Remove the heptane and most of the fixative, and replace with PBT containing 4% formaldehyde. Continue fixation for another 20 min with gentle mixing.
4. Wash 4X with PBT and procede to the Proteinase K and subsequent steps, as described above for embryos. Use the appropriate reagents for single or double FISH, or FISH: protein double-staining.
1. Template DNA should be chosen and linearized such that run-off transcripts correspond to unique portions of the gene's coding region. So far, we've found that run-off transcripts ranging from about 0.4 - 1kb work well as probes. Cutting to completion generally takes 2-4hr, and should be confirmed by agarose gel electrophoresis.
2. Removal of template with DNAseI subsequent to the transcription reaction was found to be unnecessary. Precipitation of the probe with LiCl removes most unincorporated nucleotides.
3. Previous protocols (eg. Protocol 6) used carbonate degradation to reduce the size of their RNA transcripts for increased embryo permeability. In our hands this was found to be unnecessary and in fact was usually detrimental.
4. Freshly prepared formaldehyde appears to be required with the high temperatures used for RNA hybridization. Commercially prepared formaldehyde solutions, even ultra-pure, generally yield ruptured embryos.
5. Vessel sizes used here are optimized for small collections (<250 ul settled embryos). For greater collection sizes, larger vessels should be used, keeping approximately to the same relative ratios. 50 ml Falcon tubes work well for fixing and devitillinizing settled embryo volumes from ~ 0.25 to 2 ml. Care should be used as some tubes or plastics appear to interfere with fixation and devitillinazation (Sarstedt poystyrene tubes for ex.).
6. Aqueous solution interferes with the efficiency of the subsequent devitillinazation step. This has likely occurred if the devitillinization solution is cloudy and less than 80% of embryos have moved from the interphase to the bottom of the tube. Care should be taken to minimize uptake of the lower aqueous phase when drawing up embryos from the fixative into the pipette tip. Quite often, if this occurs, the phases will separate in the tip, and the lower aqueous phase can be returned to the scintillation vial. If transfer of aqueous solution has already occurred, the devitillinaztion step can be repeated as necessary by removing as much heptane and methanol as possible, replacing with fresh heptane and methanol and shaking again.
7. The extent of proteinase K digestion is a very important consideration. In general, Proteinase K digestion enhances probe accessibility and hence the strength of the signal. However, over-digestion results in poor embryo morphology and ruptured embryos. Also, when double-labeling for proteins, Proteinase K digestion can destroy the epitope. This can be remedied by lowering the working concentration of Proteinase K as required. In fact, some in situ probes work very well with little or no Proteinase K digestion. Newly prepared Proteinase K stock solutions should be tested at several dilutions and/or digestion times. Prepare a 20 mg/ml stock of Proteinase K by dissolving in sterile water and storing at -20oC in 20- 50 ul aliquots. Repeated freeze/thaws appear to increase the activity of the enzyme.
8. The antibodies used here come lyophized. For uniformity and convenience, we resuspend the powders in 50% glycerol and then aliquot and store at -70oC. One of the aliquots can be kept at -20oC for convenience (relatively stable and unfrozen).
9. Antibodies described here have been chosen with usefulness in double-labeling in mind. The primary antibodies are whole IgGs raised in different hosts. Similarly, secondary antibodies are selected so that they are unlikely to cross react with the second primary antibody or with each other. Jackson laboratories, from whom the recommended secondary antibodies were obtained, provide information, suggestions and many products that make choosing and obtaining the appropriate antibodies relatively easy. Secondary antibodies most suitable for multiple-labeling are designated "ML". These are generally comprised of the F(ab')2 portion of IgG antibodies that recognize both heavy and light (H and L) chains of their target antibodies. ML antibodies are also preadsorbed against multiple host sera. For this reason, and because the antibodies contain light sensitive molecules, we do not bother to preadsorb them against embryos. However, if background is obtained, this may help.
Whole-mount embryo double-labeled with fluorescein labeled fushi tarazu (green) and digoxigenin-labeled wingless (red) probes.
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