RNA In Situ Hybridization of Dissected Gonads with Digoxygenin-Labeled Probes
Protocol by Ross Francis
Modified by Min-Ho Lee
In contrast with other methods using gonads affixed to slides, the various manipulations described below (gonad dissections, hybridizations, and washes) are all done in solution, either in a glass dishes (dissections) or in small glass tubes (6 mm ID x 50 mm). Washes are done by low-speed spins and all solutions (except fixatives) contain 0.1% Tween 20 to prevent gonads from sticking to glass. The key to making this work consistently is to fix dissected gonads in a combination of paraformaldehyde (3%) and glutaraldehyde (0.25%). The addition of glutaraldehyde results in lower background than fixation in paraformaldehyde alone and also makes gonads both less sticky and less susceptible to breakage. This protocol also works well for embryo in situs.
PBS: Make a 10x stock solution according to Sambrook et al. (Molecular Cloning).
PBT: 1x PBS containing 0.1% Tween 20.
Paraformaldehyde / glutaraldehyde fix:
Formaldehyde/Glutaraldehyde fixation works well for germline RNA in situs: gonads remain intact through extensive protease treatments (50 ug/ml Proteinase K for 30 min at RT) that would destroy gonads fixed in formaldehyde alone. This fixation has also been used with good results with some antibodies.
10 ml 16% formaldehyde (sealed ampules from EM Sciences #15710)
0.53 ml 25% glutaraldehyde (sealed ampules from EM Sciences #16220)
25 ml 0.2 M K2HPO4 (pH7.2)
15 ml ddH2O
Dispense in 10 ml aliquots and store at -20 C.
Proteinase K; 20 mg/ml. Aliquoted in 10 ul and stored at -80 oC. One time use/do not refreeze.
Hybridization buffer; 5X SSC, 50 % deionized Formamide, 100 ug/ml autoclaved Herring sperm DNA, 50 ug/ml Heparin and 0.1 % Tween-20. We make 50 ml, divide in 10 mls and store at -20 oC.
alkaline-phosphatase-conjugated anti-Dig (Fab2 fragment) from Roche (#1 093 274); the optimal dilution of this antibody has to be decided empirically. It varies from lot to lot. We have been using dilutions from as low as 1 : 1000 to as high as 1 : 2500.
Staining Solution: 100 mM NaCl; 5 mM MgCl2; 100 mM Tris (pH 9.5); 0.1% Tween 20; 1mM Levamisole. Levamisole is a potential inhibitor of endogenous phosphatases.
Sigma Fast BCIP/NBT tablet (#B5655); Dissolve one tablet in 10 ml staining solution. If there is a non-specific background signal, you can dilute more (up to 20 ml).
Culture tubes: Available from Fisher as 6 mm (ID) x 50 mm (length) in either Pyrex or borosilcate glass. The Pyrex version can be baked to decontaminate for RNase and also has a white area to label tubes.
Dissection dishes: Available from Carolina Biological Supply.
QIAquick PCR Purification kit from Qiagen (#28104); Works well to remove unincorporated dNTP and PCR primers.
DIG DNA Labeling Mix, 10 x conc. From Roche (#1 277 065)
Preparation of single-stranded probes from cloned cDNA
Rule of thumbs is More probe, Better Staining with less background
1) PCR amplification of the cDNA of your interest with any set of primers. We usually set up two 50 ul PCR reaction with 35 cycles. Run 3 ul each in agarose gel with any MW standard. PCR product has to be single band and has to be lot more intense than MW standard.
2) Combine two PCR reaction and use QIAquick PCR Purification kit to purify PCR product away from any unincorporated dNTP and PCR primers. Elute with 25 ul EB. The concentration should be above 200 ug/ml.
3) Set up asymmetric (one-way) PCR to generate DIG labeled probe
10x Taq Buffer 2.5 ul
DIG DNA Labeling Mix, 10 x conc. 2.5 ul
Primer * (10 pmol/ul) 1.0 ul
Purified PCR product (at least 200 ug/ml) 4.0 ul
Taq Polymerase 2 units
ddH2O to 25 ul
* Use only one primer. We usually make a sense probe with 5′-primer and an anti-sense probe with 3′-primer. RNA in situ with the sense probe will be a negative control.
4) Incubate labeling reaction for 35 cycles.
5) 75 ul of H20 is added to the reaction and 90 – 95 ul of the diluted reaction is transferred to a new tube.
6) 10 ul of 1M NaCl and 3 vols of 100% EtOH are added to the diluted reaction. After 30 min at -70 C or overnight at -20 C, the reaction is centrifuged at 15,000 rpm for 10 min. The pellet is washed in 70% ethanol, dried, and resuspended in 400 ul of hybridization buffer.
7) The probe is boiled for 1 hr. This step reduces the length of the probe for efficient penetration.
8) Optional; Probe production is assayed using the following protocol. 1 ul of probe in hybridization buffer is mixed with 5 ul of 5 x SSC, boiled for 5 min, and cooled on ice. 1 ul of this mixture is spotted on a nitrocellulose strip. Several dilutions of a pre-labelled control DNA (1ng to 1pg / ul; Boeringher Mannheim) are also spotted for comparison. The strip is baked for 30 min in a vacuum oven at 80oC, washed once in 2 x SSC, twice in PBT, and blocked for 30 minutes in PBT. The strip is then incubated for 30-60 min with AP-anti-DIG antibody diluted 1:2000 in PBT. After three 10 min washes in PBT, and two 5 min washes in staining solution, the strip is developed in staining solution with BCIP/NBT. Spots should be visible within minutes. Spot intensities of the probe and control dilutions are compared to determine the concentration of the probe.
9) Probes can be stored at -20oC for long time (I used as long as 2 years old probe without any reduction of signal).
Dissections and Fixation
1) Wearing latex gloves (optional), prepare dissected gonads (see above for dissection) and fix in 3% paraformaldehyde / 0.25% glutaraldehyde / 0.1 M K2HPO4 (pH7.2) for 2 hr at room temperature. We routinely dissect about 150 animals per each hybridization to obtain 40 or so gonads that are completely extruded, well stained, and unbroken.
2) Transfer fix solution to a 5 ml glass conical tube and spin in clinical table top centrifuge for 1 min on setting 3. (All subsequent spin washes are also for 1 min at setting 3). Discard the fixative properly and resuspend in 2-4 ml PBT. Spin as before.
3) If multiple rounds of dissections were carried out, combine different rounds into a single conical tube (5-10 ml) using a pasteur pipette. Spin, discard super, and add about 3 ml of 100% Methanol (MeOH) (MeOH is store at -20C). At this point, dissected gonads can be stored in 100% MeOH at -20 C for at least one week with no obvious problems.
Proteinase K digestion
4) Add 2-3 ml of PBT to the MeOH solution, and spin. Wash 2x more in PBT. Now split sample into 2 or more tubes (either into 5 ml conical tubes or into 6 mm x 50 mm culture tubes).
5) Using a 20 mg/ml stock of Proteinase K (PrK), make solutions of 50 ug/ml PrK in PBT. Digest gonads with PrK / PBT for 30 min at RT.
6) Wash 3x in PBT.
7) Fix 15 min in 3% paraformaldehyde / 0.25% glutaraldehyde / 0.1M K2HPO4 (pH7.2).
8) Wash 2x in PBT.
9) Incubate 15 min in PBT containing 2 mg/ml glycine / 0.1% Tween 20.
10) Wash 3x in PBT.
11) Divide gonads among several 6 x 50 mm culture tubes (Pyrex tubes are probably best as they can be baked to kill RNase), one tube per hybridzation.
12) Place gonads in 400 ul of 50% PBT / 50% hybridization buffer (HB) and incubate 5 min at 48 C (in water bath).
13) Pre-hybridize in 400 ul HB for 1 hr at 48 C.
14) Dilute boiled probe in HB to give 100 ul of solution. Try a 1:2 dilution for probes to messages of unknown abundance. Experience with gld-1 probes suggests that probe dilution is not critical — a strong, clean signal was obtained at dilutions between 1:2 and 1:5, but it began to fall off at 1:10. Probe to major sperm protein RNA could be diluted 1:20 without any fall off in signal.
15) Boil diluted probe 5 min, cool to 48 C, and add to gonads.
16) Hybridize for 12 to 36 hr at 48 C(a 36 hr hybridization may give increased signal and lower background than 15 hr). We routinely hybridize two overnight.
17) Add 400 ul of HB that is pre-warmed to 48 C, spin down, remove supernatant.
18) Wash 4 x 15 min in HB at 48 C.
19) Wash: 1 x 10 min in 50% HB / 50% PBT at 48 C
2 x 10 min in PBT at 48 C
1 x 5 min in PBT at room temperature.
20) Incubate 15 min in PBT / 0.5 mg/ml BSA (either fraction V or restriction enzyme grade).
22). Dilute alkaline-phosphatase-conjugated anti-Dig (Fab2 fragment) to optimal conc. in PBT / BSA. Add 500 ul to each tube and let go O/N at 4 C or at room temperature for 2 hr. For gonads, O/N in the refridgerator is definitely better than 2 hr at room temperature.
23) Wash at room temperature as follows:
2 x 2 min PBT
3 x 20 min PBT / BSA
2 x 5 min in Staining Buffer (see below).
24) Transfer gonads to staining buffer with BCIP/NDT and 100 ng/ml DAPI and protect from light. Signal will come up in anywhere between 5 min and 6 hr. For convenience, the reaction can be monitored by transferring tube contents to a glass dish and inspecting periodically under the dissecting scope. However, stain will appear darker in the dissecting scope than under Nomarski optics with a 40X or 100X lens.
25) Stop reaction by washing 3x in PBT. Finally place gonads in PBS containing 100 ng/ml DAPI.
Making a Mount
26) Using a drawn capillary pipette, transfer settled worms onto a large 2% agar pad that covers most of a slide. After drawing off exess liquid with a capillary, an eyelash hair can be used to push gonads and intestines away from one another. Cover with a large (24 x 50 mm) coverslip, taking care not to move the coverslip once in place. Also do not seal the coverslip immediately — image may improve as liquid evaporates and gonads become somewhat flattened. Slides can be stored at 4 C for a week or more, particularly if sealed with nail polish around the periphery of the coverslip.