[Histonet] ligand binding

[Adam Perry]

hello all,
 
I am trying to work up a protocol for detecting oxytocin receptors in rodent brain sections using a biotinylated ligand and subsequent avidin-biotin-HRP steps for visualization.  Our lab currently uses autoradiography, but I'd like to use the biotinylated method for more discrete localization.  Does anyone have experience with ligand binding studies or better yet non-isotopic binding?  If so, my specific questions are:
 
1) How does fixation of sections alter binding?  Currently we use fresh frozen brains, cut sections on cryostat and then lightly fix the sections in formaldehyde prior to binding incubation.  Is it possible to perform ligand binding on tissue perfused with 4% formaldehyde and then sectioned- I would rather work with tissue fixed before cutting- of course I'm sure different ligands have different requirements...  
 
2)  Is it necessary to fix the sections after the binding step and washes to "trap" the ligand-receptor complex for A/B development?  I've seen protocols that do both (fix or no fix after binding).
 
3) Has anyone done tyramine signal amplification in conjunction with biotinylated-ligand binding? Does it help, or not really required?
 
Any other comments on your experiences would be most appreciated.
 
Thanks in advance,
Adam Perry
Department of Physiology and Biophysics
University of Illinois at Chicago

		
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