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DESCRIPTION OF THE PROBLEM No staining SAMPLE Mouse brain (fixed frozen) PRIMARY ANTIBODY activated caspase 3 (ab2302)1:30, 1:50, 1:100 diluted in either Dako diluent or 1% BSA/0.3% triton X in PBS incubated for 1 hour at room temperature, overnight at 4C or overnight at room temperature DETECTION METHOD Fluorescence or DAB POSITIVE AND NEGATIVE CONTROLS USED Negative control: sections incubated without primary antibody Positive control: sections from mouse brain having undergone motor cortex lesion 24 hours or 5 days earlier. These sections stain positively for TUNEL. ANTIBODY STORAGE CONDITIONS Frozen in small aliquots at -20C. Aliquots are thawed and stored at 4C as needed. FIXATION OF SAMPLE formalin (perfused and post-fixed in formalin overnight) ANTIGEN RETRIEVAL I have tried boiling citrate buffer (15 minutes) PERMEABILIZATION STEP 0.3% triton X in the blocking solution (30 minutes) and in the antibody dilution solution. BLOCKING CONDITIONS I have tried commercial blocking solution, 1% BSA plus triton X in PBS, and Image-IT FX signal enhancer (30 minutes each) SECONDARY ANTIBODY anti-rabbit 488 1:500 for one hour anti-rabbit biotinylated antibody 1:300 for 30 minutes diluted in diluent or 1% BSA/0.3% triton X in PBS HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 10 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? antigen retrieval, blocking solution, concentration of primary antibody, duration and temperature of primary antibody incubation, detection method (DAB and fluorescence)
Asked on Feb 10 2012
I'm sorry to hear you are having problems with this antibody. Thank you for taking the time to fill in the questionnaire, it is very useful for me to understand your protocol. Your staining procedures and troubleshooting are excellent.
Caspase-3 activity is extremely time dependent. In example, in Jurkat cell culture, caspase activity is maximal at 6-8 hours following treatment of cells with 2 uM camptothecin. At 12-24 hours following treatment, the activity is back to the control cell level. Therefore, choosing the right time points is critical for analyzing caspases. I see that we state on the datasheet that if no signal is observed a time course may be required to identify maximal caspase activity. I see that your samples have undergone motor cortex lesion 24 hours or 5 days earlier prior to staining. Have you attemped using earlier time points with this product? I would suggest trying earlier time points on control samples.
I hope this information is helpful. Please let me know if you have any questions.
Answered on Feb 10 2012