Hi every body,
I prepared a microtisssue from autoflourescence cells, which when I did control negative flourescence staining for that (without using primary antibody) and using a redemitting signal of flourophore as secondary antibody, I see my sample showing green autoflourescenec signal (without shoing the red signal of SA) under microscope. I wanna to observe the presence specific protein (elastin) in this sample, can anybody give a solution ?
Moreover I did a staining for a specific protein, and I see nice structure but I am not sure can normal flourecence staining for a autoflourescence sample be used?
Thanks in advance
Your possibilities depends upon your equipment. You probably will use antibodies to detect proteins, and a secondary that doesn't bind the tissue. If your tissue doesn't fluoresce in the red channel, you can choose your probe here. Otherwise far red is normally out of the autofluorescent area (Cy5 e.g.). Be aware that endogenous biotin can bind SA and give unspecific signals as well, which can be blocked.
Both Vector Labs and ThermoFisher sell products to reduce background fluorescence, but I haven't tested this yet. We use the far red channel when ever we meet such problems.
Good luck!
You could also try to lower the dilution of primary and secondary antibody for your protein of interest.
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining