In our lab we have recently performed several proximity ligation assays (Duolink) in which the signals at times - but not always - distribute very unevenly across the coverslips. The unevenness of the signal can be as much as a factor 10 in which one side of the coverslip has little signal and the other side a very strong signal. It's quite puzzling as the cellular distribution is completely homogenous and the treatment is identical.
Has anyone else come across this, and do you have any ideas as to what might have happened?
Yes! It happened to me more then once. I normally put two brain sections on a slide. The sections are sequential, so they shouldn't vary in their protein expression and still I see different PLA signal in both sections. I use KO brain sections as a negative control. At some point I started thinking that whoever is cutting the brain is trying to 'mess' with me and he/she puts wt and ko sections on one slide... Have no idea how to overcome this obstacle.. Did you?
In our case it appeared to be a problem with partial desiccation during incubation as we were using a modified protocol. Try to see if there's any point in your protocol where desiccation may potentially be occurring, even for short periods of time. Let me know if it changes anything!
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