My tissue is a mouse tissue and my primary antibody also mouse. I have already tried mouse on mouse kit. ı have still staining at negative control. My tissue have parafin sections.
Can anyone help me solve about this ?
Dear Kübra,
besides the most useful suggestion by Sebastian Schmitt I would try an antibody from a different species.
Best regards
Daniela
Dear friend,
You should prepare fresh %5 BSA blocking agent and as more intense.
ı have already tried a protocal about %5 BSA blocking, it had beeen blocking before primer antibody and seconder antibody 1 hour at room temparature.
my negative control had still staining .
Maybe it will help you ... http://www.abcam.com/protocols/mouse-on-mouse-staining-protocol
Some very useful reagents from Dako include the Dual Peroxidase Block reagent, their antibody diluent, and their protein block. They work perfect with my tissue sections and the antibodies I am working with. I would also suggest GBI (Golden Bridge Inc.), they have cool kits that I use and every kit is specific to the kind of tissue (human, mouse, rat,...) and the type of antibody you are testing. You should not have any background staining with your negative control. That means that your antibody is binding to something in your tissue, or you have not properly blocked non-specific binding. Dont forget to perform antigen retrieval. Citrate Buffer works very well with most tissues and antibodies. I can send you my protocol if you send me your email. Good Luck!
Dear Dr Ayman, you can reduce the non specific back ground by increasing the time of peroxidase block eith using the protein block like serum is species that the secondary antibody was synthesized and more washing grade by washing buffer particularly after steps if secondary antibody
Dear Kübra Aksu,
First of all, I suggest to determine the source of the background like antibodies or inappropriate fixation. In case the background related to antibodies, you can use lower concentration or decease incubation time or increase washing time.
I also recommend to have positive and negative controls that could help you in this regard and you can also try appropriate blocking solution.
Good luck
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