Antibody staining with Anti-MAPK 

Try Anti-MAPK antibody in Membranipora cleavages, Terebratalia gastrula, and Lineus cleavage and also Tyrosinated tubulin in Terebratalia larva as control.

We split the sample to develop with DAB/HRP protocol and fluorescent.

Anti-MAPK

Membranipora

Cleavages

Anti-MAPK

Terebratalia

Gastrula

Anti-MAPK

Lineus viridis

Cleavage

Tyrosinated tubulin

Terebratalia

Larvae

Primary antibodies

Monoclonal Anti-MAP Kinase, Activated (Diphosphorylated ERK-1&2) antibody produced in mouse
Suggested dilution: 1:1000 (~0.5-1 μg/mL, see specs).
Observations: @Henry2008 used 1:200 (~30 µg/mL) with a different antibody; @Koop2007 used a final concentration of ~3 µg/mL. @Amiel2013 used 1:200, but does not specify which antibody was used.
Final concentration we used: 30 µg/mL.

Tyrosinated Tubulin (Mouse) 1:250.

Secondary antibodies

  • Anti-Mouse (Goat) HRP + POD (Jackson) → 1:250 each! (1 µL for each of the tubes we have)
  • Anti-Mouse (Goat) AlexaFluor 594 → 1:250.

Protocol

We always used 0.3% PTx except for Membranipora (0.1% PTx) with 500 µL washes.

1st day

  • 5x 5 min washes in PTx.
  • 1x 30 min in PTx.
  • 1x 1h in PTx.
  • 2x 15 min in PTx+BSA.
  • 1x 30 min in PTx+NGS.

Incubated with Anti-MAPK 30 µg/mL and Tyrosinated tubulin 1:250 at 17h30.

2nd day 06/06/13

  • 3x 5 min PTx+BSA
  • 5x 30 min PTx+BSA
  • 1x 30 min PTx+NGS

Samples were split to 2 dishes and incubated with secondary antibodies as described above.

3rd day 07/06/13

DAB-HRP reaction (DAB powder borrowed from S4).

  1. Used similar buffer as for in situ: TBS = 20 mM Tris, 500 mM NaCl, pH 7.5.
  2. Dissolve 50 mg DAB in 100 ml TBS. Add 10 μL 30% H2O2. Make this solution just before use.
  3. Remove PTx+BSA and add DAB+H2O2 solution to well. Staining comes immediately, we left for 6 min and it became overdeveloped in the larvae. In Membranipora it was fine.
  4. Stopped the reaction with 2x 5 min washes of PBS (protocol recommends distilled H2O).

Terebratalia larvae and Membranipora 32 cell embryo worked! We lost fluorescent control and Lineus and Terebratalia gastrula showed no staining, except for what it looked to be unfertilized eggs from Terebratalia.

Mmem_DAPI Mmem_MAPK