Whole mount in situ hybridization

(Probe)
1. Linearize 10 µg of plasmid DNA by digesting with a suitable enzyme in 100 µl scale.
2. Check for complete digestion by running 1 µl of DNA solution.
3. Do Isopropanol precipitation following Phenol/Chloroform treatment.
4. Dilute precipitated DNA with 25 µl of water (for 4 uses).
5. Mix 6 µl of lineaged DNA solution, 2 µl of 10 x Transcriptional buffer, 2 µl of labelling mixture, 1 µl of RNA gurad, and 1 µl of RNA polymerase (T3, T7, or SP6?).
6. Incubate mixture at 37 ºC for 2 h.
7. Remove unicorporated, free nucleotides with Quick Spin Columns:
How to use Quick Spin Columns.
1) Remove caps from columns (top first!!)
2) Spin for 5 minutes (4 ºC, 1.8 krpm) in a swing bucket centrifuge.
3) Remove the eluate and spin again.
4) Put columns in new tubes, add transcription reaction making sure to add it directly on resin while not disrupting resin with the pipet tip.
5) Spin for 15 min (4 ºC, 1.8 krpm) in a swing bucket centrifuge
6) Run 1µl on a gel to quantify the RNA yield and to determine how much to use for the in situ hybridization.
7) Store at -20ºC. Use 1-5 µl of probes for hybridization (depend on the quality of probes).


(Sample)
1. Fix albino embryos in MEMFA for 1h at RT or overnight at 4 ºC. 2. (Replace with PBS for 5 min at RT) x 2. 3. (Replace with MtOH) x 2 and store at -20 ºC.

(1st Day)

Note. ALL steps in Day 1 are done ON ICE except for the Proteinase K treatment (step #3).
Note. Each wash, unless indicated, is with at least 2mls of buffer.
Note. Prepare 4% paraformaldehyde/0.2% glutaraldehyde in fresh PBS: dissolve paraformaldehyde in PBS and heat at 60C, mixing occasionally until completely dissolved, cool on ice, filter, and then add glutaraldehyde. About 5mls will be need for each sample (step #6).
Note. Have hybridization solution ready - aliquoted and stored at -20 ºC.

1. Rehydrate the embryos through a methanol series in PBSw (75%, 50%, 25%) incubate each step for 5 minutes.
2. Wash 3 times with PBSw, 5 minutes each wash.
3. Digest embryos with 10ug/ml Proteinase K in PBSw at room temperature (1ml per tube). Staining for highly expressed genes requires less digestion, but longer digestion may help for genes with lower expression. In general use: Frog embryos 8 minutes.
4. Stop digestion by washing with 2mg/ml glycine in PBSw.
5. Do a fast wash with PBSw, and then wash 2 times with PBSw, 5 minutes each wash.
6. Refix embryos in 5mls of 4% paraformaldehyde/0.2% glutaraldehyde in PBSw for 15 minutes.
7. Do a fast wash with PBSw, and then wash 3 times with PBSw, 5 minutes each wash.
8. Wash in 1ml of 50%PBSw:50% hybridization solution for 3 minutes.
9. Wash in 1 ml of hybridization solution (100%) for 3 minutes.
E Embryos can also be stored at this step in hybridization solution at -20 ºC.
10. Replace 1ml hybridization solution with 900 µl hybridization solution and prehybridize for 3 hours at 65 ºC.
11. Denature 200ng of probe in 100 µl hybridization solution at 95C for 5 minutes. Add this probe/hybridization mix to the embryos (final concentration of 200ng/ml), and hybridize overnight at 70 ºC.

(2nd Day)

Note. Prepare Antibody Buffer V enough for 2.5ml/vial.
Note. a) Dissolve 10% Boehringer blocking reagent in maleic acid buffer (MAB). Heat and vortex frequently to dissolve completely. Store at -20 ºC as a stock solution.
b) To make Antibody Buffer: 10% heat inactivated goat serum, 10% Boehringer blocking stock solution from Step a, 80% PBSw, Heat at 70C for 10 minutes, vortex, cool on ice and filter.


1. Remove probe/hybridization mix and replace with 800 µl Hybridization Solution. Wash for 5 minutes at 70 ºC.
2. ADD 400 µl 2x SSC, pH4.5 to each vial. Incubate 5 minutes at 70 ºC. Repeat the addition 2 more times (final volume = 800 µl + 400 µl + 400 µl + 400 µl = 2 ml), incubating at 70 ºC for 5 minutes each time.
3. Remove the mix and wash with 2x SSC, pH7/0.1% CHAPS at 70 ºC for 30 minutes. Repeat this wash.
4. Wash 2 times in MAB at room temperature for 10 minutes each wash.
5. Wash 2 times in MAB at 70 ºC for 30 minutes each wash.
6. Wash 2 times in PBS at room temperature for 10 minutes each wash.
7. Wash in PBSw at RT for 5 minutes.
8. Incubate the embryos in 1ml Antibody Buffer (without antibody) at 4 ºC, rocking, for at least 2 hours. At this time also pre-block the antibody in remaining Antibody Buffer, with rocking at 4 ºC for 2 hours.
Anti-Dig-Alkaline Phosphatase dilute 1:10,000 from a stock of 150units/200ul.
9. Replace Antibody Buffer (without antibody) with 1.5 ml of pre-blocked antibody in Antibody Buffer. Incubate with rocking at 4 ºC overnight.

(3rd Day)
1. Fast wash embryos with 0.1% BSA in PBSw.
2. Wash 5 times in 0.1% BSA in PBSw, with rocking, 1 hour each wash at RT.
3. Wash 2 times in PBSw for 30 minutes each wash, at RT.
4. Wash 2 times in AP1 Buffer for BM Purple staining for 10 minutes each wash.
5. Replace AP1 Buffer with 1ml BM Purple, cover with foil and incubate with rocking until desired staining is reached. Staining time will vary depending on the level of expression. The reaction will go faster at room temperature, but when at 4 ºC, the embryos tend to get less background.
6. Stop staining reaction by washing thoroughly in PBSw. -OR- Wash 2 times in Stop Solution for 15 minutes each wash. Dehydrate through a methanol series (25%, 50%, 75%, 2x 100%), and store in methanol at
-20 ºC.
 Solutions:
10x PBS: 80g NaCl, 2g KCl, 14.4g Na2HPO4, 2.4g KH2PO4, pH to 7.4, up to 1L by DDW, DEPC treat and autoclave.

PBSw: PBS with 0.1% Tween-20

20x SSC: 175.3g NaCl, 88.2g Sodium Citrate, pH to 7.0, up to 1L by DDW, DEPC treat and autoclave.

Hybridization Solution: Make 1L, filter, and store at V20 ºC in aliquots.
(1st step) 10g Boehringer Block, 500ml Formamide, 250ml 20x SSC, Heat at 65 ºC for 1 hour
(2nd step) 120ml DEPC treated water, 100ml Torula RNA (10mg/ml in water; filtered), 2ml Heparin (50mg/ml in 1x SSC), 5ml 20% Tween-20, 10ml 10% CHAPS, 10ml 0.5M EDTA.

MAB: 100mM Maleic Acid, 150mM NaCl, pH 7.5

Antibody Buffer: 10% Heat Inactivated Goat Serum,1% Boehringer Block, 0.1% Tween-20 Dissolve in PBS at 70 ºC, vortexing frequently, and then filter (0.45 µm).

AP buffer: Put 5 ml of 1M NaCl, 1M Tris, pH 9.5 and 5 ml of 0.5M MgCl2 into 40 ml of DDW.
Note. Do not mix at high concentration, or prepicitate appear.

Stop Solution: 100mM Tris pH7.4, 1mM EDTA

Product:
Boehringer Block - Roche #1096176
Proteinase K - Gibco #25530-049
Anti-Dig-AP - Roche #1093274
BM purple - Roche