The cells are EndoC-BH4. They are grown on matrigel, but they do not seem to be submerged into the gel. I tried to stain them with antibodies, phalloidin, and DAPI after paraformaldehyde fixation and permeabilization. The outcome was very poor. Has anybody a good experience with any cells grown on matrigel? Any advises?
By cells grown on matrigel, do you mean you coat the plate with matrigel before seeding your cells on top and let them grow on the coated plate? The standard protocol for immunofluorescence should works nicely. I do not see anything special about growing cells on matrigel that requires special treatment for staining. Furthermore, if you mention you stain DAPI, practically any cell containing DNA will stain them if you permeabilize them well. You may have problem with the concentration you use that requires further optimization.
Thank you. In fact, i did not expect problems, too. It did not turn well, however.
What are you using to permeabilize? I'd try 0.5% Triton/PBS for 15 mins, wash and stain incubating for a longer period of time and/or at a higher concentration of DAPI/phalloidin. I'd expect at least one of these will show an improvement from your first round of staining and you can optimize from there.
- Badges
- Science topic
- Similar topics
- Gels