Dear scientific community,
I faced an unexpected problem regarding the detection of a protein in the murine muscle stem cells. I am interested in a GPCR, which has a relatively low mRNA expression (RNA-Seq). Anyway, on a couple of immunostainings I was able to detect it. I assume that the IF worked correctly, also the secondary AB only (Alexa 594) showed no specific signal at all.
To make life easier, and also for live-cell imaging, I ordered a transgenic mice, which has a hrGFP reporter sequence at the end of the last exon of the GPCR of interest (global knock-in). The mouse is published already, and I also detected the hrGFP signal in the positive-control tissue.
But I was quite surprised that the hrGFP signal was not detectable/on the level of autofluorescense in the muscle stem cells, that I am interested in, even though I saw the clear signal with the IF.
Have anyone experienced such situation, when a reporter signal is much lower than the signal of IF? What would you suggest to try?
Thanks in advance!
Best,
Michail