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I'm having trouble with this antibody as it is giving me a lot in terms of what I believe to be non-specific staining, cells fluorescing (colocalize with DAPI) in 5um sections of parrifin embedded, formalin fixed, brain sections of naive mice, but they are not neurons (do not colocalize with non-specific neuronal marker). Have there been any complaints of impurities in this particular lot that may be causing this reaction? We have attemtpted caspase 3 before, but in mouse not rat and have been successful. I've tried several different protocols, any ideas of what is going on? Again, there is reactivity, but in naive mice as well. blocking condition: 10% goat/1% BSA 20-60 minutes (tried, 20, 40, 60) permeabilization with TX-100 0.3% 60 minutes detection: tried LSAB and fluorescence (alexa 488) antigen retrieval (heat based sodium citrate) 2ndary (alexa 488, 1:250-1:1000 tried) 1ary (1:20-1:100 tried) wash: PBS 5x3 min between antibodies parafin embeded sections, fixed with PFA: brain sample
Asked on Jun 29 2012
Thank you for sending your protocol details. We have not received other complaints about this lot, but I do see one complaint from February about a different lot but with a different issue: no staining at all.
If I understand you correctly, you expect to see activated caspase 3 in neurons, in both groups of mice, naive and treated. Do you have any other evidence that the treatment is inducing apoptosis in neurons? Can you please send an image of the stain?
Are you confident your secondary antibody and reagents are not the source of the stain? A no-primary-antibody control will confirm, if this is the case.
I look forward to your reply.
Answered on Jun 29 2012