Hello Scientific Community,
I need your help, I did an immunostaining of P-gp on Caco-2/TC7 cell line. I used a classic protocol with 4% PFA fixation and without permeabilization. You can attached a picture of my immunostaining. this does not resemble what I have observed on other immunostaining targeting P-gp. First only few cells are stained. Second, it's look like a staining with permeabilization protocol. Third, I find the stringy aspect very strange.
My hypothesis is that only the dead cells were labeled which explains why it looks like labeling with permeabilization the filamentous aspect perhaps representing cell fragmentation
maybe you have another hypothesis on what I observe?
PS: The control with the secondary antibody alone shows no staining
Thank you for your response and your time,
Maxime Dubois, PhD Student
It is possible that the P-gp in your cells was denatured during fixation with 4% PFA, which can lead to a lack of antigen recognition and low staining intensity. It is also possible that the fixation process disrupted the structure of the cells leading to the filamentous appearance. Another possibility is that the antibody you used for the immunostaining is not specific enough for P-gp and is binding to non-specific sites on the cells (if not tested already). I would recommend trying a different fixation method or using a different antibody to see if that resolves the issue.
Sudarshan Bhattacharjee Thank you for your answer, i will try a different protocol for fixation, antibodies used have already been tested.
Hello Khin Thuzar Aung
thank you for your answer, No nucleus are stained with Hoescht, and yes it works without permeabilization.- Badges
- Science method