[Histonet] re: phosphate buffered saline or Tris PBS ?

[PW Howorth, Physiology]

Hi,

I'm currently using immunohistochemistry (free floating sections or 
serially mounted sections) to map the rat brain.  We use viral injections 
to express green fluorescent protein and use 1 or 2 other labels (i.e. Cy3 
or AMCA) to colocalise receptors with GFP expression.

I have been trying to improve our methods that we currently use. 
Originally we used Tris PBS (0.01M, pH 7.4), I notice that other groups use 
a variety of differences in these recipes, what advantages does Tris PBS 
offer (if any) or would you recommend using PBS (either 0.1M or 0.01M) ?

Secondly, when using triton x-100 (0.3%) has anyone noticed a reduction 
with GFP signal compared to just using 50% ethanol ?

And finally, we use cardiac perfusion to initially fix tissue followed by 2 
hours post fixation with 4% phosphate buffered (0.1M, pH 7.4) formalin. 
The antibody we use for mu opioid receptors (diasorin) - does anyone have 
any experience with this antibody and this method have you done antigen 
retrieval to improve the signal ?

Many thanks

Patrick

----------------------
Patrick Howorth,
Dept of Physiology,
University of Bristol,
Bristol,
BS8 1TD.
+44 (0)117 33 17112
patrick.howorth <@t> bristol.ac.uk