MAPK Membranipora immuno


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Incubated Membranipora 10h embryos with MAPK antibody. Will leave it for 2-3d.

06/09/13

Washes with PBT and added Anti-Mouse POD (Jackson) + Anti-Mouse HRP 1:250 each. Incubated in the cold room.

07/09/13

Selected a few embryos to do the TSA amplification step only.

  1. Washed out secondary with PBT (3x5min and 4x30min).
  2. Transferred embryos to a 0.5 mL eppendorf and removed most of the liquid.
  3. Diluted DNP stock solution 1:50 in 1x amplification buffer from the kit and added to the tube for 5 min.
  4. Stopped the reaction with the Detergent Solution 2x 5 min at RT.
  5. Inactivated POD just to be sure incubating embryos in 0.1% H2O2 in PTx for 30 min at RT.
  6. Blocked 40min with 5% NGS in PTx.
  7. Incubate with Anti-DNP-HRP overnight 1:100 in 5% NGS at 4 °C.

08/09/13

  • Wash with PBT 4x 15 min.
  • Wash with PTx 4x 30 min.

09/09/13

  • TSA amplification step 1 µL of Cy5 + 49 µL amplification reagent.
  • Stop with 60 °C detergent solution + 1x detergent solution at RT.
  • Wash 3x with PTx.
  • Incubate with 1.25U of Phallacidin for 2h. After 1h added DAPI dilution to final volume of 1:1000 (250 Phall. + 50 µL of DAPI).
  • Washed samples 3x with PBS.
  • Confocal time!

Detergent washes

Apparently it does not make much difference of doing the detergent wash at 60°C or just PTx washes. Maybe the detergent hurt the phallacidin?

DNP+TSA

Doing the DNP amplification step before the TSA results in a lot of signal in the egg shell, which is not that good for imaging.

TSA only

Only the TSA seems to be enough for a stable signal and not so much background.