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?
I was involved in some HRP tract tracing experiments in the CNS a few years
ago. We used Wheatgerm Agglutinin-HRP, with a similar injection protocol
to you, or alternatively small (5-50nl) pressure injections. Our results
were very good.
Also, we perfused with freshly made 4% (W/V) paraformaldehyde, followed
by the same volume of 10-20% sucrose PBS, then store in 30%PBS for a day
or two.
So, maybe you could try WGA-HRP, or perhaps the preservatives in the formalin
may affect the HRP, so try paraformaldehyde.
There are different TMB reaction methods, but they all should work quite
well.
Feel free to email me directly if you want to discuss the technique in
more detail
best of luck,
Jaimie Polson
Department of Physiology, F13
University of Sydney
Australia
