I am performing IHC using mouse brain slices. I am performing double labeling and the blocking buffer I am using is normal goat serum. One target remains constant in my experiments, and the second target is using an antibody against a specific protein and the other is against a ribosomal RNA. The first (constant) target is raised in guinea pig. The second targets are both mouse monoclonal. The secondary for the first target is donkey anti-guinea pig, and for the second target(s) goat-anti mouse. When I perform double labeling using the first target and the RNA target I have no problem with cross reactivity, the signals are separate (so everything is fine). Now the problem occurs when I try looking at the first target with the second target (which is now a protein) I get identical signals. I ran no first target (but with the first target secondary) with second target and there is no cross reactivity, I only see a signal in the channel for the second target. When I do the opposite control I only see a signal for the first target. The weird thing is when I add both primaries and secondaries together now I am seeing complete overlap. These data suggest that the primaries are somehow recognizing each other. Can anyone help me understand what is going on here? Any suggestions? If you need additional info, just ask. Thank you!
Hi Matthew,
Is there any chance that your two protein targets are actually co-localized within the cell? If so, maybe your results are fine. If that isn't something that you would expect or that would make sense based on the target proteins, then have both of the primary antibodies been validated in your lab previously? If you run a Western blot using each primary antibody, does it recognize a target protein of the right size in a nice, clean band?
~Sarah
Hey Sarah, The co-localization is too exact, and when I run my second target alone the staining looks nothing like it does when both primaries are added together. To answer your second question: No they have never been used in my lab, and I have no personally validated them. However these are well cited in the literature. Furthermore, when I run target one with the RNA target this is not a problem.
Hi Matthew, do you mean that with both primaries the staining of the second target looks exactly like the normal staining for the first? If yes, have you ever tried staining sequentially? First against the second target and then against the first? That should solve the problem, no?
Hope that helps
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