On Thu, 25 May 2000, Colleen Forster wrote:

> I am reposting 2 questions that I have received no replies on.
 
> 1. Is anyone doing GABBA staining? 

      If you got no replies, it looks as if nobody is! 
      I've not done this myself but have read about it,
      so here are some musings, mostly about your Point 2.

> 2. After profusing with 80% alcohol for 20 minutes I drop mouse
> brains into fresh 80%. How long will they need to post fix in
> alcohol before processing?
 
      If I remember rightly, the papers on immunohistochemistry
      of amino acid neurotransmitters (glutamate, GABA, glycine)
      in the 2nd half of the 1980s used glutaraldehyde-containing
      fixatives and frozen or vibratome sections - the latter for
      subsequent electron microscopy. A small molecule like GABA
      would be dissolved out by water or alcohol. Glutaraldehyde,
      however, can combine with the amino group of the amino acid
      and with an amino group of a nearby protein molecule. This
      anchors the small molecule to the fixed tissue. 

      The antibodies to these amino acids (which are not, of
      course, normally immunogenic) are made by inoculating
      animals with an a protein-glutaraldehyde-neurotransmitter
      conjugate. The resulting antisera include antibodies that
      recognize the amino acid-glutaraldehyde-tissue complex. 

      I don't think it will be possible to immunostain for
      GABA in alcohol-fixed tissue. You might be able to detect
      glutamate decarboxylase, an enzyme involved in the
      biosynthesis of GABA. Enzymes are proteins and as such 
      are coagulated by alcohol, which sometimes improves the
      exposure of antigenic sites, The general structural
      preservation won't be very good after 80% alcohol with
      no other ingredients. Many fixative mixtures that are 
      excellent for brain are mostly alcohol, but the other
      ingredients (especially acetic acid) make important
      contributions too. Specimens are usually fixed by
      immersion in these liquids. Perfusion of alcohol is
      likely to clog up the blood vessels by insolubilizing
      the blood (or the salt, if you wash out the blood with
      saline).

      If it would help, I could post to Histonet some references
      to papers on GABA immunohistochemistry. Just ask. You
      could also try a PubMed search, which would probably pick
      up more recent articles than the ones I've made notes
      about. A firm called Chemicon sells antibodies to amino
      acids, and they surely must test them. I don't have their
      plump catalogue to hand now, so can't quote, but it
      probably contains good advice. (The thickness of the
      Chemicon catalog has increased X4 or X5 in the past 12
      years or so.)

      The development of methods to localize free amino
      acids and other small molecules using antibodies was a
      notable achievement of 1980s histochemistry. Perhaps in
      the 2000s we'll see the unequivocal and selective 
      localization of acetylcholine. This was the first 
      neurotransmitter to be identified, back in the 1930s,
      some 20 years after its discovery as a physiologically
      important compound. 

      End of transmission.

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1