In <D0x821.L14 at cunews.carleton.ca> rlabelle at dag.carleton.ca (Rosalie Labelle) writes:
>Want small localized HRP injections but I'm having problems with poor
>transfer (poor labelling of cell bodies). Procedure has entailed
>giving extracellular iontophoretic injections (5 microAmperes positive
>alternating current for 10 min.) of Sigma 6 HRP (20% w/v in distilled
>water). Following 2 day transfer time, perfuse with 0.9% saline, fix
>with 10% formalin, rinse with 0.9% saline 1 hour later. Slice and
>store in cryoprotectant (sucrose, PVP, ethylene glycol). Wash with PBS
>and react with TMB method.
>What am I doing wrong?
Your procedure sounds fine. It could however be the HRP. In our
lab we use Sigma Type IX and maybe XII. We also only allow a 24hr uptake
period. This works consistently. The only problems we have ever had was
with the HRP itself.
Jonathan Byrd
Dept. of Neuroscience
University of Rochester
Rochester, NY, USA
