In situ Hybridization for Xenopus Embryos

(Modified from protocol of Carole Labonne, Northwestern University)

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; 100l of 5mg/ml ProteinaseK, 50l of 20KU/ml Hylaurinodase, and 500l 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; 100l 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; 125l 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; 250l 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 500l hybridization buffer, shake at 60C for 10 minutes.

11.           Replace it with fresh 500l hybridization buffer, shake at 60C for 1 hour.

12.           Replace it with fresh Probe solution

q      500l hybridization buffer with 1l probe.

q      Hybridize by shaking overnight at 60C


Day 2 (total time 5 hours):

1.              Replace Probe solution with 500l of hybridization buffer.  Shake at 60C for 10 min

2.              Rinse:

q      3 x 20 min with 2X SSC at 60C

q      2 x 30 min with 0.2X SSC at 60C

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 4C

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 4C

q      Prepare fresh; 10ml 5XMAB/5XBMB, 10 Heat-treated lamb serum, 30 ml ddH2O and 18l 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 4C 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 335l of NBT and 175l 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 4C.

q      Reaction may be accelerated by incubation at 37C.

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 4C.

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 -20C.

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.