In Situ Hybridization to Dissected Gonads with Digoxygenin-Labeled Probes

 

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 backgound 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.

Dissections and Fixation

1) Wearing latex gloves, prepare dissected gonads and fix in 3% paraformaldehyde / 0.25% glutaraldehyde / 0.09 M K₂ HPO₄ (pH7.2) (see below) for 2-3 hr at room temperature. I always do several rounds of dissections (which are later combined in one tube) in order to obtain enough material for several hybridizations. You should start with several hundred dissections per hybridization in order 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 2. (All subsequent spin washes are also for 1 min at setting 2). Discard fix and resuspend in 2-4 ml PBTw. 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 -20°). At this point, dissected gonads can be stored in 100% MeOH at -20° for at least one week with no obvious problems.

Proteinase K digestion

4) Add 2-3 ml of PBTw to the MeOH solution, and spin. Wash 2x more in PBTw. 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 10 mg/ml stock of Proteinase K (PrK), make solutions of 25 ug/ml and 40 ug/ml PrK in PBTw. Divide the gonads in half. Digest one half of the gonads in 3 ml of 25 ug/ml PrK / PBTw for 30 min and the other half in 3 ml of 40 ug/ml PrK / PBTw for 30 min.

6) Wash 3x in PBTw.

7) Fix 15 min in 3% paraformaldehyde / 0.25% glutaraldehyde / 0.1M K₂ HPO₄ (pH7.2).

8) Wash 2x in PBTw.

9) Incubate 15 min in PBTw containing 2 mg/ml glycine / 0.1% Tween 20.

10) Wash 3x in PBTw.

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.

Hybridization

12) Place gonads in 400 ul of 50% PBTw / 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°.

14) Single-stranded DNA probes are made by asymmetric PCR using a single primer (see Patel and Goodman protocol distributed by G. Seydoux).

15) Dilute boiled probe in HB to give 100 ul of solution. Try a 1:3 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.

15a) Boil diluted probe 5 min, cool to 48°, and add to gonads.

16) Hybridize for 12 to 36 hr at 48° (a 36 hr hybridization may give increased signal and lower background than 15 hr).

17) Draw off and save hybridization solution. Wash 2 x 2 min in HB at 48°.

18) Wash 4 x 30 min in HB at 48°.

 

19) Wash:

1 x 10 min in 50% HB / 50% PBTw at 48° 2 x 10 min in PBTw at 48° 1 x 5 min in PBTw at room temperature.

20) Incubate 15 min in PBTw / 0.5 mg/ml BSA (either fraction V or restriction enzyme grade).

22). Dilute alkaline-phosphatase-conjugated anti-Dig (Fab2 fragment) to 1:2500 in PBTw / 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 PBTw
3 x 20 min PBTw / BSA
2 x 5 min in Staining Buffer (see below).

24) Transfer gonads to staining buffer containing 4.5 ul NBT/ml, 3.5 ul X-phosphate/ml 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 PBTw. 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° for a week or more, particularly if sealed with nail polish around the periphery of the coverslip.

 

Materials

Most of the solutions are from Geraldine Seydoux's and Andy Fire's in situ protocol for embryos. Most solutions are DEPC-treated. The two reagents from Boeringer Manheim (10x dNTPS with digoxygenin dUTP and affinity-purified anti-Dig antibody) can be purchased indvidually or as part of a more expensive kit.

PBS: Make a 10x stock solution according to Sambrook et al. (Molecular Cloning ). DEPC treat and autoclave.

PBTw: 1x PBS containing 0.1% Tween 20.

Paraformaldehyde / glutaraldehyde fix:
10 ml 16% formaldehyde (sealed ampoules from EM Sciences)
0.53 ml 25% glutaraldehyde (sealed ampoules from EM Sciences)
43 ml 0.1 M K₂ HPO₄ (pH7.2)

Dispense in 10 ml aliquots and store at -20°.

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.

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.