Hi everyone,
I am performing some immunofluorescence in mice's muscle tissue. I am doing some antibodies concentration testing and the thing is:
When I put my tissue under microscope, there is no exaggerated brightness at first, but I have a slight marking that appears more when I increase the gamma and gain parameters.
Question: is this ok? I mean... can I change these parameter until I can see my marking more brilliantly, or can this be manipulation?
(It is noteworthy that there are no new "markings" when I do this, only the ones that exist get brighter.)
Thanks!
Hi Garciana
You can change the parameters to increase brightness. It just means either the antibody is weakly binding or the flourophore is not too strong. This is not manipulation as long as you apply the same parameters for negative control (no antibody).
cheers
Dharanija
Hello, Mr.
We can change the parameters to increase brightness to show the connective tissue then knew the contain of the muscle by using the
fluorescence microscope.
Hi Graciana,
Both laser power and Gain power will influence your fluorescent intensity, I will check my secondary antibody control (without adding primary antibody) first, make sure you cannot see the signal from a normal working power of laser and gain and then use the same setting to look at other samples. In this case you are confident that the signal you collected is real signal rather than background from seconday antibodies. Good luck for the experment.
Cheers,
Chenxi