Whole Mount Non-radioactive In Situ Hybridization On Mouse Embryos

• Page Contents:
• Tissue Preparation
• ISH - day 1
• ISH - day 2
• ISH - day 3
• Stock Solutions

Tissue Preparation

Materials needed:

• Baked glass or sterile plastic petri dishes
• Dissection tools
• 4% Paraformaldehyde in PBS (PFA, make from 8% PFA and 10x PBS stocks)
• *50% Methanol in PBS (make with DEPC H₂O, MeOH and 10x PBS stock))
• *100% Methanol

* RNAse free

Procedure:

1. Anesthetize pregnant dam and remove uterus to petri dish containing 4% PFA.

2. Dissect embryos away from uterus and extraembryonic membranes in a separate dish containing 4% PFA. Embryo staging (crown-rump length determination) and dissection of specific tissues may be done at this step. Place each embryo, or piece thereof, in an autoclaved 1.5 ml microcentrifuge tube containing 4% PFA. Older embryos (³ E16) may require larger containers.

Josh’s numbering system: ‘date of sacrifice’.’animal # for that day’ ‘embryo letter’ So, 990123.2A would be embryo ‘A’ from the second litter taken on January 23, 1999. Embryos are assigned a letter by starting at the base of the uterus and alternating sides as you count back towards each uterine horn. Thus, a higher letter indicates a more distal position in the uterus. An eight-embryo litter might be designated as follows:

3. Fix embryos 20 minutes-overnight in 4% PFA at 4° C. Shorter fixation times may improve detection of weak signals. Embryo staging and dissection of specific tissues may be done at this step.

4. If long term storage of tissue is not needed, begin day 1 ISH procedure at step #2. Otherwise, wash tissue 5 min. in 50% methanol in PBS.

5. Wash tissue 2x5 min. in 100% methanol and then replace the 100% methanol one final time. Tissue may be stored up to 2 years at -20° C in 100% methanol. DO NOT dissect embryos/tissue while in methanol.

In Situ Hybridization

DAY 1

ALL OF DAY 1 IS PERFORMED USING RNASE FREE TECHNIQUE

Materials Needed:

• Plastic or baked glass transfer pipettes
• *50% methanol in PBS
• *PBST: 1x PBS (made from 10x stock) containing 0.1% Tween-20 (1:100 dilution of 10% Tween-20)
• 10 mg/ml Proteinase K
• 4% PFA in PBS
• *Hybridization mix:
  • 4.5 ml deionized formamide (aliquots at -20° C)
  • 2.25 ml 20x hybridization salts
  • 90 µl 100x Denhardt’s solution
  • 180 µl 10% Tween-20
  • 225 µl DEPC H₂O
• *10 mg/ml Salmon sperm DNA (aliquots at -20° C)
• *10 mg/ml poly A RNA (aliquots at -20° C)
• *DEPC H₂0
• *50% Dextran Sulfate

* RNAse free

Procedure:

Note: All washes and incubations on all days are performed at room temperature with gentle agitation unless otherwise specified.

1. Wash tissue (stored in 100% MeOH at -20° C) in 50% methanol for 5 minutes.

2. Wash tissue 4x5 min. in PBST. During the third wash, specific tissues may be dissected from the embryos.

3. Incubate tissues in 10-20 µg/ml Proteinase K (1:1000 dilution of stock solution) in PBST for 15-30 minutes at room temperature. Example times I use are as follows:

4. Wash tissue 2x10 min. in PBST

5. Refix tissue in 4% PFA in PBS for 20 min. Specific tissues may be dissected out at this time. If multiple embryos/pieces will be assayed by the same probe they may be combined in the same tube at this step.

6. Wash 2x10 min. in PBST

7. While the last washes are going, prepare the prehybridization buffer.

1. For each 1 ml of prehyb buffer needed, combine:
   • 25 µl Salmon Sperm DNA stock
   • 25 µl Poly A RNA stock
   • 145 µl DEPC H₂O.
2. Boil this mixture 3 min., then place on ice 1-2 min. Combine with 805 µl of hybridization mix to yield 1 ml of prehybridization buffer.

8. Place a suitable amount of prehybridization buffer in each microfuge tube to completely cover the tissue and prehybridize 1-2 hours at 42° C. This is an emergency stopping point. Tissue may be stored for a few days at -20° C in prehyb buffer either before or after prehybridization.

9. Towards the end of the prehybridization, prepare the hybridization buffer.

1. Calculate the volume of hybridization buffer that will be needed for each probe. (One tongue needs 20-50 µl of fluid to be covered)
2. Next, make probe mixture by combining:
   • 1/40 volume Salmon sperm DNA (final concentration 250 µg/ml)
   • 1/40 volume Poly A RNA (final concentration 250 µg/ml)
   • RNA probe (final concentration 0.2-0.5 µg/ml) - typically 1/50 volume
   • DEPC water to make this mixture 8.5% of hybridization volume
3. Boil this probe mixture 3 min. and place on ice >1-2 min.
4. Prepare hybridization-dextran mixture (hyb-dex) by adding 110 µl 50% dextran sulfate to 890 µl hybridization mixture for each ml of hyb-dex needed.
5. Add the appropriate amount of hyb-dex to the probe mixture (91.5% of hybridization volume) to arrive at the proper volume of hybridization buffer.

10. Replace the prehybridization buffer with the hybridization buffer and hybridize overnight at 60° C.

Note: SSC solutions for tomorrow can be prepared now and placed in 60° C water bath.

DAY 2

Materials needed:

• 2x SSC (1:10 of 20x SSC stock)
• 0.2x SSC (1:100 of 20x stock)
• 0.1 M Phosphate buffer (PB, 1:2 of 0.2 M stock)
• 1:1 mix of 0.2x SSC and 0.1 M PB
• Blocking solution (for 10 ml)
  • 10 % normal sheep serum (1 ml)
  • 4% dry milk (optional) (0.4 g)
  • 2 mg/ml Bovine Serum Albumin( 200 µl 100 mg/ml stock)
  • 0.3% Triton X-100 (1 ml 3% stock)
  • in 0.1 M PB (5 ml 0.2 M PB)
  • (2.8 ml dH₂O)
• Anti-digoxigenin, Alkaline Phosphatase-conjugated Fab fragments (Boehringer)

Procedure:

Note: RNAse free technique is not important after the hybridization step.

1. Wash tissue 3x10 min. in 2x SSC at 60° C.

2. Wash tissue 3x 10 min. in 0.2x SSC at 60° C.

3. Wash tissue 10 min. in 0.2x SSC/0.1 M PB.

4. Wash tissue 2x 10 min. in 0.1 M PB.

5. Incubate tissue in blocking solution 1-2 hours.

6. Incubate tissue overnight at 4° C in blocking solution containing a 1:1000 dilution of anti-digAP antibody. Incubation may go over 3 nights (1 weekend), but reduce antibody concentration to 1:2000 in this case.

DAY 3

Materials needed:

• 0.1 M PB
• 0.1 M Tris, pH 7.4 (1:10 of 1 M stock)
• Coloration buffer (for 100 ml)
  • 0.1 M Tris, pH 9.5 (10 ml 1M Tris, pH 9.5)
  • 50 mM MgCl₂ (1.01 g MgCL₂•6H₂O)
  • 100 mM NaCl (2 ml 5 M NaCl)
  • 0.1% Tween-20 (1 ml 10% Tween-20)
  • dH₂O to 100 ml
• NBT and BCIP stock solutions (Boehringer)
• 4% PFA in PB or PBS

Procedure:

1. Wash tissue 2x10 min. in 0.1 M PB.

2. Wash tissue 10 min. in 0.1 M Tris, pH 7.4. Tissue should be transferred to the wells of a 24 well culture dish at this point.

3. Wash 2x10 min. in coloration buffer.

4. During step 3, prepare coloration reaction buffer by adding 3.5 µl BCIP stock and 4.5 µl NBT stock to each 1 ml of buffer needed (0.5-1 ml/well depending on the size and amount of tissue in each well).

5. Incubate tissue in coloration reaction buffer for 15 minutes to 2 hours in the dark. Buffer may be replaced if reaction seems to have slowed after 45-60 min. My reactions typically go 30 min. and I "call it off" after an hour.

6. Wash tissue twice in 0.1 M PB. If coloration is too light, coloration can be redone by returning to step 3.

7. Refix and store tissue in 4% PFA.

8. Photograph tissue in PB or PBS.

Created 5/25/1999. Last saved 05/25/1999 2:18 PM.

Stock Solutions

• DEPC H₂O
  Add 500 µl DEPC per 1L dH₂O. Shake, let stand 1hr, autoclave 20 min. liquid cycle.
  
  
• 8% Paraformaldehyde (100 ml)
  Heat ~80 ml H₂O to 60° C. Add 8.0 g Paraformaldehyde. Turn off heat and stir briskly. Add 1-2 drops 10 N NaOH and stir until clear. Filter through #1 filter paper. Bring up to 100 ml. Store at 4° C.
  
  
• 10x PBS (1L)
  • 9.94 g Na₂HPO₄ (dibasic) anhydrous
  • 3.60 g NaH₂PO₄ (monobasic) anhydrous
  • 75.972 g NaCl
  Add to 800 ml DEPC H₂O. Bring up to 1 L and check pH is 7.4. Add 500 µl DEPC, shake, let sit 1 hr, autoclave 20 minutes liquid cycle.
  
  
• 10% Tween-20 (100 ml)
  Add 10 ml Tween-20 to ~85 ml DEPC H₂O and bring up to 100 ml. Stir to dissolve detergent.
  
  
• 20x hybridization salts (for 100 ml)
  • 3 M NaCl (17.53 g)
  • 100 mM EDTA (3.72 g)
  • 100 mM PIPES, pH 6.8 (3.02 g)
  Heat to 60° C and pH to 6.8 to get into solution. Bring to 100 ml with DEPC treated dH₂O. Add 50 µl DEPC, shake, let sit, autoclave.
  
  
• 100x Denhardt’s (for 50 ml)
  • 2% polyvinyl pyrrolidone (1 g)
  • 2% Ficoll (1 g)
  • 2% Bovine serum albumin (1 g)
  Bring to 100 ml with DEPC H₂O, aliquot, and store at -20° C
  
  
• 50% Dextran Sulfate (50 ml)
  To 25 g dextran sulfate, add DEPC H₂O to 50 ml. Dissolve by shaking (this takes a while.) Store at 4° C
  
  
• 20x SSC (1 L)
  • 175.3 g NaCl
  • 88.2 g Sodium Citrate (citric acid)
  Add to 800 ml ddH₂O. Adjust pH to 7.0 with NaOH. Bring up to 1 L. Add 500 µl DEPC, shake, let sit, then autoclave
  
  
• 0.2 M Phosphate Buffer (2 L)
  
  • 42.6 g Na₂HPO₄ (dibasic) anhydrous
  • 13.8 g NaH₂PO₄ (monobasic) anhydrous
  Add to 1800 ml dH₂O and bring up to 2 L. pH to 7.2.
  
  
• 100 mg/ml Bovine Serum Albumin
  Add 100 mg BSA to 1 ml dH₂O in microfuge tube. Vortex to dissolve. Store at 4° C.
  
  
• 3% Triton X-100 (100 ml)
  Add 3 ml Triton X-100 to ~95 ml dH₂O. Stir to dissolve. Store at 4° C.
  
  
• 1 M Tris, pH 7.4 (1L)
  • 140.4 g Tris HCl
  • 13.4 g Tris base
  Add to ~850 ml dH₂O. Adjust pH to 7.4. Bring up to 1 L.
  
  
• 5 M NaCl (1 L)
  
  Add 292.2 g NaCl to ~800 ml DEPC H₂O and dissolve. Bring up to 1 L. Add 500 µl DEPC, shake, let sit, then autoclave
  
  
• 1 M Tris, pH 9.5 (500 ml)
  • 7.0 g Tris HCl
  • 55.4 g Tris base
  Add to ~300 ml dH₂O. Adjust pH to exactly 9.5. Bring up to 500 ml.