hi
I want to detect GluR2 (subtype of AMPA reseptor) by ihc staning, but i don,t knew how to manage the process of it
please help me how to doing this protocol step by step?
thanks for your attention
These articles may help,
1. Evidence for low GluR2 AMPA receptor subunit expression at synapses in the rat basolateral amygdala. Divina S. Gryder, Dora C. Castaneda, Michael A. Rogawski, DOI: 10.1111/j.1471-4159.2005.03334.xV
2. Age-Related Loss of the AMPA Receptor Subunits GluR2/3 in the Human Nucleus Basalis of Meynert. Milos D. Ikonomovic, Experimental Neurology166, 363–375 (2000) doi:10.1006/exnr.2000.7544,
See protocol described our paper:
Pickard L, Noël J, Henley JM, Collingridge GL, Molnar E (2000) Developmental changes in synaptic AMPA and NMDA receptor distribution and AMPA receptor subunit composition in living hippocampal neurons. J Neurosci 20:7922-7931.
Hi Ghasem, you may get more specific answers if you elaborate on what it is exactly you're looking to do - IHC, I gather that's in tissue? Is it brain sections, spinal cord...etc? Or is it in cell culture?
Hello,
The answer is not as simple as one may believe.
What do you want to do?
As far as I know, Glur2 are mainly expressed at synaptic sites. If you want to specifically quantify synaptic receptors you should not use a simple approach such as IHC. Unless you work on cultures, synaptic receptors are not easily accessible from their extracellular side for staining because they are embedded in the PSD. In these conditions IHC would mainly label GLUR2 receptors located in the neuron cytoplasm.
You should therefore use antigen retrieval (pepsine, microwaves) to allow antibodies to reach Glur2 from the extracellular space.
See:
Fritschy JM, Weinmann O, Wenzel A, Benke D. Synapse-specific localization of NMDA and GABA(A) receptor subunits revealed by antigen-retrieval immunohistochemistry. J Comp Neurol. 1998;390:194–210.
: Balland B, Lachamp P, Kessler JP, Tell F. Silent synapses in developing rat
nucleus tractus solitarii have AMPA receptors. J Neurosci. 2008 Apr
30;28(18):4624-34. doi: 10.1523/JNEUROSCI.5355-07.2008. PubMed PMID: 18448639.
2: Balland B, Lachamp P, Strube C, Kessler JP, Tell F. Glutamatergic synapses in
the rat nucleus tractus solitarii develop by direct insertion of
calcium-impermeable AMPA receptors and without activation of NMDA receptors. J
Physiol. 2006 Jul 1;574(Pt 1):245-61. Epub 2006 May 11. PubMed PMID: 16690712;
PubMed Central PMCID: PMC1817785.
3: Lachamp P, Balland B, Tell F, Baude A, Strube C, Crest M, Kessler JP. Early
expression of AMPA receptors and lack of NMDA receptors in developing rat
climbing fibre synapses. J Physiol. 2005 May 1;564(Pt 3):751-63. Epub 2005 Feb
24. PubMed PMID: 15731186; PubMed Central PMCID: PMC1464465.
Thanks for all your answers.
Hello Sir Altimimi. I want to detect GluR2 subtype of AMPA receptor in postsinaptic membrane cells in hippocampous region with paraffin embedded sections.
Hello and thanks Mr. Fabien tell. My aim of this research is not count of AMPA receptor, but meagerment of optical density neurons react by using software imageJ.
Thanks for your attention.
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