Membranipora MAPK staining on in situ embryos

Selected 5 genes for a MAPK immuno staining to resolve the spatial relationship between MAPK vegetal blastomere, gene expression and the embryonic axes.

gata_b foxa
six3/6 evx
nk2.1

I started to de-glycerol embryos in hourly washes of PTw around 1200.

20/08/2015

  • 3x 10min PTx
  • 2x 45 PBT
  • 2x 30min PTx+NGS
  • 1:200 anti-mapk antibody added at 14:00

21/08/2015

  • Wash primary antibody (afternoon)
  • 3x 5min.
  • 4x 30min.
  • Add secondary antibody anti-mouse pod at 1:250 dilution at 1800.

22/08/2015

  • 3x 5min PBT washes.
  • 4x 30min PBT washes.
  • Overnight at 4°C in PBT.

23/08/2015

  • Washed 3x 5min in TNT buffer.
  • Developed in 50 µL TSA + Cy5 fluorochrome for 3 min.
  • Washed 10min in detergent solution at 67°C and another with warm detergent but at room temp to cool down.
  • Washed 2x 5min PBS.
  • TDE series ending with 97% TDE in PBS with DAPI 1:1000.

24/08/2015

Mounted slides and checked under scope. There is quite some background with the orange filter for cy3. I cannot distinguish any signal at the posterior vegetal blastomere. Confocal showed that there was no signal from the MAPK antibody. Maybe the longer time in glycerol messed the epitopes? Or the time developing (3min) was not enough…

DAPI (cyan), MAPK (magenta), in situ (yellow):