Question (35963) | Anti-Cleaved Caspase-3 antibody (ab2302)

DESCRIPTION OF THE PROBLEM No staining SAMPLE Mouse brain (fixed frozen) PRIMARY ANTIBODY ab2303, diluted in dako diluent or 1% BSA/0.3% triton X/PBS, diluted 1:30, 1:50 or 1:100, incubated at room temperature for one hour or overnight at 4C or at room temperature. DETECTION METHOD fluorescence or DAB POSITIVE AND NEGATIVE CONTROLS USED Negative: no primary antibody Positive: brain sections from mice that underwent motor cortex lesion 1 or 5 days before perfusion. These sections show positive TUNEL staining. ANTIBODY STORAGE CONDITIONS Stored in small aliquots at -20C. Aliquots are thawed and stored at 4C as needed. FIXATION OF SAMPLE formalin (perfused then post-fixed overnight) ANTIGEN RETRIEVAL Boiling citrate buffer for 15 minutes PERMEABILIZATION STEP 30 minutes in 0.3% triton X (triton X is also included in the antibody dilution solution) BLOCKING CONDITIONS Dako blocking solution or ImageIT FX signal enhancer or 1% BSA/0.3% triton X in PBS SECONDARY ANTIBODY anti-rabbit Alexa 488 (1:500 in dako diluent or 1% BSA/0.3% triton X/PBS for one hour at RT) or anti-rabbit biotinylated IgG (1:300 in Dako diluent or 1% BSA/0.3% triton X/PBS for 30 minutes at RT) 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, concentration of primary antibody, duration and temperature of primary antibody incubation, detection method