Re: Immunohistochemistry is making me cry

From:Todd Sherman


Based on your current body of evidence, it would appear that your Guinea 
Pig x Rat mu-op-rec1 pAb is specific and effective, depending on the 
morphology of the tissue.  The most important component of your 
procedure (primary Ab) is OK and you report that the protocol does work. 
So, the problem seems to be duplicating the results in disparate 
"supportive" tissue rather than modifying the IHC-detection protocol per se.

I hope I'm not stating the obvious or exposing my ignorance but... can 
you not duplicate the tissue preservation/fixation process for the brain 
sections that you used for the spinal cord and myenteric plexus? 
Forget, for the moment, prepping beautiful morphology of the brain 
tissue. Can you prepare a small piece of brain in the exact same manner 
as the other CNS tissues where you have previously experienced positive 
anti-mu immunoreactivity?  If you can, then can you get "ugly" brain 
sections that are immunoreactive?  If the immunoreactivities in all of 
the CNS are similar at this point, then your dilemma can be narrowed 
down to the preparation of large brain sections for immunoreactivity. 
If you cannot get similar immunoreactivity in all CNS tissues, then the 
problem might be more severe.

Until you know that this particular pAb works under a specific fixation 
process, your results might not be adequately interpretable.  You know 
that your anti-mu protocol works for some tissue but now you may be 
trying to extrapolate those results to a tissue that has been prepared 
differently.  Do not be tempted to be led down that path unless 
absolutely necessary, and even then, have a prepared disclaimer.

Or, you may have discovered that mu opioid receptor-1 is not as abundant 
in a particular rat species of a particular age after all.  That might 
be another part of your proposal to the extended scholarship... but I 


Todd Sherman
HistoSoft Corporation
"Biology in a new form..."

 >---------------------------major snippage------------------------------
 >Unfortunately the co-localisation aspect is an absolute linchpin of my
 >work;  this is an all or nothing situation.   To add insult to injury,
 >the mu Ab works brilliantly well in the spinal cord and fairly well in
 >the myenteric plexus.
 >Andrew Gray
 >PhD student

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