Re: GABA-staining

From:John Kiernan

Dear Luz Cardenas and Histonetters,

GABA differs from your other three antigens in being
a small, soluble molecule (an amino acid). The other
three are peptides or proteins, with bigger molecules.

Immunohistochemical localization of the amino acid
neurotransmitters depends on their immobilization by
rapid cross linking to adjacent protein molecules.
The antisera and antibodies that recognize GABA are 
raised in animals inoculated with GABA-protein 
conjugates. The GABA is a hapten; it changes the
shape of the associated protein and makes it look
foreign (not self) to the immune system of the
inoculated animal. The resulting antibodies bind 
to larger molecular formations (epitopes) whose
shapes are determined by the inclusion of chemically
bound GABA. 

If you didn't fix your specimens in a mixture that
contained glutaraldehyde, they probably contain
no GABA. 

Here's a list of references. The first two will 
probably be the most helpful. They are the classics
in the field of GABA immunohistochemistry, and need
to be read thoroughly. I hope the abbreviated format 
looks OK in the email. This is the first time I've 
posted references in this way to Histonet. 
You should see nine neat one-liners.

Hodgson,&c    1985  Antisera to -aminobutyri J
Histochem Cy 33:229-23
Somogyi,&c    1985  Antisera to -aminobutyri J
Histochem Cy 33:240-24
Jessen,&c     1987  GABA as an autonomic neur Trends
Neurosc 10,:255-2
Augustine     1987  Immunocytochemical staini Brain
Res      424:352-3
Bannerman,&c  1987  Analysis of enteric neuro
Developmental  9,:201-22
Denizot,&c    1988  The use of Quetol 651 for Histochem
J    20:222-22
Ohara         1988  Synaptic organization of  Journal
of Ele 10,:283-2
Crooks,&c     1992  Localization of GABA, gly J Comp
Neurol  315:287-3
Charara,&c    1996  Glutamatergic inputs from J Comp
Neurol  364:254-2
On my screen the symbol for the gamma in GABA looks
like a French 
c with a cedille or the equivalent letter in Brazilian
The journal names are truncated, but are still obvious,
and the tail
ends of the page numbers are all gone. Perhaps this
isn't the best
way to give library directions!  If you like it let me
know. If you
don't, say nothing and I won't do it again. 
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
Luz Cardenas wrote:
> Hello all,
> I am doing 4 Immuno-staining: Enkephalin, GFAP, Tyrosine Hydroxylase and GABA.
> Doing the 4 at the same time, on the same brain, with the same protocol. I got stain in 3 of the 4.
> Do some body have an idea why I don't get GABA staining at all? (we did diferents dilutions). Is something special with GABA?.
> Thank you very much.
> GABA is from Chemicon.
> Luz Cardenas, MD
> Post-doctoral Research Trainee
> Department of Anatomy and Neurobiology
> University of Tennessee Health Science Center
> 855 Monroe Avenue, Room 309/302
> Memphis, TN 38163


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