i am currently doing ICC for Pax7(mouse) and MyoD(rabbit) within a slide chamber.
My concentration for:
- Pax 7 primary Ab is 1:15 to 1:100 and secondary is alexa fluor 546(1:300)
- MyoD primary Ab is 1:15 to 1:100 as well, secondary is donkey anti rabbit biotinylated(1:300) and tertiary is alexa fluor 488 strepavidin conjugated 488(1:400).
Blocker i am using is 5% goat & donkey serum in PBS with 0.35% carrageen.
the thing is MyoD signals comes out fine..however pax7 i can never get it to show..Neg control shows nothing...i have not tried positive controls.
any idea what is going wrong??
would appreciate is someone can help