In article <1994Dec17.120427.3240 at galileo.cc.rochester.edu>
jb019a at uhura.cc.rochester.edu (Jonathan David Byrd) 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?
>>Water may be the wrong vehicle. Need to dilute HRP in buffered-saline,
and keep pH such that HRP has positive charge (I think 7.4 will work).
Also, 5.0 uA seems high for HRP, 2 uA is more common. Good luck
>> Doug Fitzpatrick
