Day 1 (total time 3 hours 30 min):
1. Rehydrate embryos with 5 min washes of:
q 75% Ethanol & 25% ddH2O
q 50% Ethanol & 50% ddH2O
q 25% Ethanol & 75% PTW
q 100% PTW (3 x 5min)
2. Add Devitellinization Solution for 10 min.
q Prepare fresh; 100µl of 5mg/ml ProteinaseK, 50µl of 20KU/ml Hylaurinodase, and 500µl of 200mg/ml Collagenase A in 50ml of PTW
q Do not leave embryos in solution for more than 10 min
3. Add proteinase K solution for 5 min.
q Prepare fresh; 100µl of 5mg/ml ProteinaseK in 50ml of PTW
q Do not leave embryos in solution for more than 5 min.
4. Rinse 2 x 5 min with 0.1M TEO
q Do not leave embryos in solution for more than 5 min.
5. Add TEO solution (TEO #1) and nutate for 5 min.
q Prepare TEO #1 fresh; 125µl of Acetic Anhydride in 50ml of 0.1M TEO
q Do not leave embryos in solution for more than 5 min.
6. Pull off half of TEO #1 and add more concentrated TEO #2, nutate for 5 min.
q Prepare TEO #2 fresh; 250µl of Acetic Anhydride in 50ml of 0.1M TEO
q Do not leave embryos in solution for more than 5 min.
7. Wash 2 x 5 min in PTW on the nutator.
8. Refix for 20 min in MEMFA
q Prepare this solution fresh
q 5ml of 10X MEM and 5ml of 37% Formaldehyde to 50ml ddH20
9. Rinse 3 x 5 min in PTW.
10. Add 500µl hybridization buffer, shake at 60åC for 10 minutes.
11. Replace it with fresh 500µl hybridization buffer, shake at 60åC for 1 hour.
12. Replace it with fresh Probe solution
q 500µl hybridization buffer with 1µl probe.
q Hybridize by shaking overnight at 60åC
Day 2 (total time 5 hours):
1. Replace Probe solution with 500µl of hybridization buffer. Shake at 60åC for 10 min
2. Rinse:
q 3 x 20 min with 2X SSC at 60åC
q 2 x 30 min with 0.2X SSC at 60åC
q 2 x 15 min with 1X MAB at RT
3. Add 1:5 5XMAB/5XBMB/ddH2O blocking solution, nutate for 1 hour at RT.
q Prepare fresh; 8ml 5XMAB/5XBMB and 40ml ddH2O
4. Add 2:2:6 5XMAB/5XBMB/Heat-treated lamb serum/ddH2O. Nutate for 1 hour at RT or overnight at 4åC
q Prepare fresh; 10ml 5XMAB/5XBMB, 10 Heat-treated lamb serum and 30 ml ddH2O
5. Add 2:2:6 5XMAB/5XBMB/Heat-treated lamb serum/ddH2O containing a 1:3000 dilution of the affinity-purified sheep anti-DIG-AP antibody. Nutate for 4 hour at RT or overnight at 4åC
q Prepare fresh; 10ml 5XMAB/5XBMB, 10 Heat-treated lamb serum, 30 ml ddH2O and 18µl affinity-purified sheep anti-DIG-AP antibody
Day 3 (total time 5 hours):
1. Wash the embryos 5 x 1 hour each at RT with 1X MAB.
q One of the washes can be overnight at 4åC if begun on Day 2.
2. Wash the embryos 2 x 5 min at RT with Alkaline Phosphatese (AP) Buffer.
q Prepare AP buffer fresh every time.
Stock |
Final |
For 50 ml |
1M Tris (pH 9.5) |
100mM |
5.00 ml |
1M MgCl2 |
50mM |
2.50 ml |
4M NaCl |
100mM |
1.25 ml |
10% Tween-20 (depc) |
0.1% |
0.50 ml |
Levamsiole |
1mM |
0.012 g |
q Add ddH2O upto 50ml
3. Replace AP buffer with 335µl of NBT and 175µl of BCIP in 50ml of AP buffer. Incubate and nutate at RT in dark.
q Check every 30 minutes for signal for 2 hours
q Reaction may be slowed down by incubation at 4åC.
q Reaction may be accelerated by incubation at 37åC.
4. Stop the chromogenic reaction when satisfied with signal and background by replacing the AP buffer with MEMFA for 1 hour at RT or overnight at 4åC.
q Prepare this solution fresh
q 5ml of 10X MEM and 5ml of 37% Formaldehyde to 50ml ddH20
5. Wash with 1X MAB for 5 min
q Steps 5-7 are required only if bleaching pigmented embryos
6. Bleach embryos
q Bleach in light box checking every 30 minutes.
q Prepare this solution fresh
q 5ml Formamide, 1ml 20X SSC, and 1.67ml H2O2 (30%) to 50ml ddH2O.
7. Wash 2 x 5 min with 1X MAB
8. If background is high, embryos maybe incubated in a light box with a methanol and H2O2 (70:30, respectively) solution for no more than 1 hour.
9. Replace solution with ethanol and store in glass vials at -20åC.
q Embryos maybe made transparent/translucent by placing them in a 2:1 benzyl benzoate/benzyl alcohol solution as to facilitate analysis of certain deep tissue markers
q Pictures should be taken as soon as possible since ethanol will decrease signal over time.