The tracer has a biotin tag on it. So you need to attache this to an Avidin which has a HRP label attached to it.  This is done by the "ABC" complex=The avidin-  biotinylated- HRP complex. This is what will  attach to the DBA. This complex is what is found in the Vector stain kit.  When you mix this solution up , you must let it complex for a few minutes and then put it on your sections. Are your sections free floating? If so how thick? If they are over 50 mics, you might consider incubating them over night. Where they stored in formalin? If so they must be washed well in buffer, as formalin inhibits the HRP reaction. Are the processes you are looking at fine? Then you may consider amplifying the signal. Did you  put your  sections on slides before the reaction? If you used gelatin or albumin to attach them, this can block access of the ABC. Free floating is recommended.  Did you incubated in the DAB for the right length of time? You should check under the scope (care being taken as DAB is toxic!).

There are a number of paper you can see, I cit only one, See:  REINER a. ET AL  J of Neuroscience methods 2000, 15;103(1);23-37 Good luck

Thank you, Frank

We have tried the ABC kit, with varying time frames of DAB incubation following the kit. I have used HRP/DAB staining many times in the past and the fact that we haven't seen anything resulting from the ABC kit OR a streptavidin solution surprises me. I'm starting to think that perhaps we may have an issue with the BDA tracer itself (not sure exactly what the issue is, but we're ordering another kit to see if we can get it to work better). 

All sections are free-floating, and were stored in a buffer solution (not stored in formalin). The processes are definitely fine, and I have inspected the region they should be in under 63x oil immersion, and I do not see any, which is one reason why I am thinking we may have an issue with the tracer itself, as opposed to the staining procedure). 

You mentioned something about albumin...we have an adapted protocol for our ABC/streptavidin incubation that includes a blocking step using bovine serum albumin. Do you think this may have something to do with not getting a stain? Essentially, we do a 0.75% H2O2 incubation, followed by washes, followed by a 5% NDS, 3% BSA, and 0.2% triton PBS blocking solution, then we move the tissue from that incubation solution directly into either the ABC or into the 1:5000 streptavidin solution (depending on which way we want to attach the HRP). after doing this, we have no stain once processed with DAB. Thoughts?

Did you include sections of your injection sight? You know you will have some of the label there and should be able to visualize it. If you do not see it, it would indeed suggest problems with the label. (either it is bad or it did not come out of the needle). The vector instructions note that solutions can be mixed with histochemical grade BSA, so I don't think that is the problem. But if it drys with the section on the slide, it could block entry of reagents. You are doing free floating so no problem with that. Good luck

Thanks again! Yes, we looked at section that had injection site...not labeling, but there is evidence of where the needle went. I agree, something might be wrong with tracer.

To eliminate the possibility that there is a complete detection failure with DAB procedures, consider localising the injection site with a fluorescently conjugated avidin or streptavidin. You may also consider including a fluorescent marker in with the BDA in the pipette next time. However, I agree that the most likely issue is that the BDA did not leave the electrode.