Membranipora MAPK immuno staining

I will test whether the MAPK antibody works after in situ hybridization and if it works after U0126 treatment.

[empty] MAPK after nk2.1 in situ
MAPK 8h 14°C control MAPK 8h 14°C U0126 treated

For the in situ well:

  • 3x 10min PTx
  • 2x 45min PBT
  • 2x 30min PTx+NGS

For the other wells I did longer PTx initial washes (45min total) and shorter PBT and NGS (as in the protocol, 2x 15min PBT, 30min NGS).

Incubated all at 4°C with 1:200 dilution of anti-mapk (new aliquot from S8).

07/04/15

Washed off primary antibody with PBT 3x5min 4x30min. Added secondary anti-mouse pod at 1:250 dilution.

08/04/15

Washed secondary with PBT washes as above and developed with 50µL of TSA solution with 1 µL of Cy5 fluorochrome. Embryos were washed 3x5min in PTx and last one for 1h. Results:

  • MAPK immuno staining works after in situ. Signal is weaker and seems to be restricted to the nuclei (cytoplasm signal unclear). The MAPK active cell is located opposite to the expression of nk2.1, which is anterior/ventral. Thus, it seems likely that the vegetal cell with MAPK activity is at the posterior/dorsal side. Plan appropriate in situs to confirm this result.
  • Controls worked ok.
  • U0126 10µM treated embryos showed no detectable MAPK activity. One embryo might had a brighter nuclei, but I need to confirm this under the confocal. Another issue is that these samples had the membrane. MAPK antibody works fine through the membrane, but maybe is better to remove them to have a cleaner image and avoid doubt.

There was some extra background, I’ll wash the embryos with detergent solution to remove some.

09/04/15

Made 30min at 67°C detergent washes (total 3x 10min). Background improved a bit, but nothing extreme.

Also did a Murray’s Clear preparation and they get completely transparent. However, the morphology is not quite optimal and embryos get fragile. Not sure if worth it.