I analyzed supposedly MSC by flow cytometry and immunofluorescent staining with 2 different CD73 antibodies. As MSC the cells are supposed to be positive for CD73.
The immunostaining shows a nice positive staining, the control (only secondary antibody) is negative. I do not have a negative sample, on which I could test the staining.
In flow cytometry, the cells are negative. The day before, other cells showed positivity with the same antibody.
Which result can I trust in? Is there any way to properly test the primary IF antibody without having a negative sample? Are there CD73 negative MSC?
Thank you Carl! We had an isotype control in flow cytometry - regarding to it, the negativity is correct. As far as I know, our antigen (CD73) is a plasma membrane protein, so I should be able to detect it without permeabilization. Can you maybe send me a protocol for permeabilization in FC, I would like to try it anyways. Thank you very much!
Hi, sorry for my intromission. Could you please send me the protocol that has worked for you on immunostaining?
I have tried culturing MSCs on coverslips, fixating them with 4% PFA, and diluting primary antibodies conjugated with FITC at 1:100 and 1:25 on BSA (1%). Until now, nothing has worked for me and nothing was seen in inverted microscope (with the properly filter). I would greatly appreciate if you could help me with some advices.
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