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HRP tract tracing advice required.

doug doug
Tue Dec 20 23:52:28 EST 1994


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




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