I'd like to fluorescently stain for activation of GABAergic neurons in the mPFC- but have a few questions.
1) When looking at the mPFC, would it be best to use antibodies for FOS and GABA, or FOS and GAD67? What are the advantages or disadvantages to each?
2) Are there any particular antibodies you would recommend for this?
3) Any tips and tricks as far as staining goes? TBS or PBS? Dilutions?
Thank you in advance!
Hi Sara,
I would use FOS and GAD67 antibodies. GAD67 will label all the cells that are GABAergic wheres, GABA will also label the vesicles and axon terminals that contain GABA not just the cells. For antibodies I would check Journal of Comparative Neurology Antibody Database at:
http://onlinelibrary.wiley.com/journal/10.1002/%28ISSN%291096-9861/homepage/jcn_antibody_database.htm
The rest should be just standard double-labelling immunohistochemistry.
Best wishes, Refik
Hi Sara,
As said by Kanjhan, you can use cFOS and GAD67 antibodies together for GABAergic neurons. You can also verify your results using cFOS and VGAT antibodies. VGAT (vesicular GABA transporter), is also specific for GABAergic neurons. To give further strength to your results, I would suggest doing triple labeling your sections using cFOS, GAD67, and either GABA or VGAT antibodies.
Antibodies for all these markers are easily available; you can check Chemicon, Santa-Cruz and also Abcam.
Best luck
Khan
1. Immunocytochemistry for GABA or the GABA synthesizing enzyme, glutamic acid decarboxylase (GAD65/67), or by in situ hybridization for GAD65/67 mRNA.
2. mouse anti-glutamic acid decarboxylase-6 (GAD-6; 1:75; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA).or Mouse monoclonal anti-GAD67 antibody (Sigma-Aldrich, G5419), diluted 1:60K (Kim et al., PLOS1 (2012)).
Sara,
A tip I can give you is to use relatively fresh tissue, meaning don't wait too long between the perfusion and slicing of the brains and the IF of cFos. In my experience, tissue older than 1 month post-perfusion had problematic cFos staining.
Dear Sara,
There was a discussion here on RG about a year ago about different cFos antibodies in which you may be interested:
https://www.researchgate.net/post/Why_do_I_get_no_c-fos_labelling_in_my_immunofluorescence_experiment/1
Refik's suggestion to look at the database is great, as is Boaz's suggestion to use freshly perfused tissue.
Good Luck!
Jill
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