I had a weird IF result and I would like to know what do you guys think about it.
First of all, I transiently transfect my cells (HEK293) with a plasmid and treated them with a peptide (10uM). My plasmid has a HA tag, so I worked Mouse-anti-HA plus anti-mouse RHO. My peptide was marked with anti-peptide + anti-rabbit FITC. The images were analyzed by fluorescent microscopy with deconvolution.
The result: I had only FITC positive cells when the cells were transfected. Cells alone didn't internalize the peptide. But the weird thing is the spots. There is too much colocalization, which make me think about cross linking Ac.reaction.
So, antibody controls were made and I observed a little bkg only in the peptide + 1* (anti-ha and peptide) and 2* (anti-mouse and rabbit) condition (Image shown above).
What can be happening here?
Thank you in advance!