Hi, I was trying to stain serotonin in my tissue. The primary antibody I used was rat-anti mouse and the secondary was goat anti-rat. For the negative control, I only put secondary antibody and I did not expect to see any signal. However, the image shows that the experimental condition have the exact signal with the negative control. I am not too sure if it is the problem with the secondary antibody or the blocking step.
Hello Serena, from which species is your tissue? Is it rat tissues which you like to investigate? The reasonis is your primary antibody because when you detect the primary ab out of rat and you use an anti -rat secondary you stain all the serum components of the rat section. That's why your negative control looks like your experimental section. I recommand in that case, use a primary antibody from any other species.
If you are working with mouse tissue you need a mouse absorbed secondary antibody.
The 3rd rason could be, indeed, the blocking step. Here you have to test systematically which part of your blocking and dilution medium are responsible for this result. On recommandtion: if you use BSA or fat dry milk, replace it with normal serum (blocking step 10%, dilution medium 1%). Use the normal serum from that species in which your secondary ab is generated, in your case from Goat. Good luck!
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