I have an E-Cadherin antibody. I have been playing around with the parameters for ICC on 293T and NIH3T3 cells.
For some reason, i keep getting a lot of nuclear localisation even if i don't permeabilise the membrane . I get what appears to be membrane bound staining as well with the 293T cells (not a great deal as i expected). The NIH3T3 cells is only nuclear.
Does anyone have any tips on how to prevent background nuclear staining?
Thanks,
Nathan
Hi William,
I have blocked with 4% PFA.
I have only tried 1 and 2% BSA blocking on its own at room temperature for 30min and 1 hr and 4 degrees overnight. I have added Tween and Triton as well but that was obviously not a great outcome.
The Primary antibody is conjugated to a Fluorophore.
I know there is a lot more to try, but desperate to see if i can get a quick solution as i need to get this sorted pretty quickly.
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining