I'm using biotinylated dextran amine (BDA) as an anterograde tracer in rats that have SCIs. I injected it into the primary motor cortex (2 injection sites, 1 ul each) and I'm having trouble seeing it in the spinal cord. I'm using tissue rostral to the injury for optimization. I can easily see the axons in the white matter of the dorsal column but I cannot see anything in the grey matter. What I do see in the grey matter after staining with streptavidin594 is splotchy and nonspecific. I've changed my concentration of strep, used BSA instead of normal serum in my block, used an avidin/biotin blocking buffer and still, nothing. I also tried strep:AF660 just in case the channel was the issue. My protocol involves a 30 min blocking step and 2 hour RT incubation with strep on free floating sections (30um). The concentrations of strep I've used is 1:1000, 1:500 and 1:250. Any advice? Should i switch to chromogenic staining (DAB, VIP, etc).

Thank you for any help!