Studies that have compared the relative distributions of GAD65 vs GAD67 have found that antibodies that primarily recognize GAD65 show enrichment at synaptic sites, suggesting that this form is primary localized there. If you are using an antibody that recognizes both forms, it is likely you would have widespread punctate staining at GABAergic synapses which may make it difficult to identify GAD-positive somata.

I have found that Abs directed against only GAD67 (such as Millipore MAB5406) reveal somata better. Others have used brain injections of colchicine to inhibit axonal transport an increase somatic labeling of GAD67 and/or GAD65.

http://www.jneurosci.org/content/14/3/1834.full.pdf

http://www.ncbi.nlm.nih.gov/pubmed/25058170

Hello

Look at papers here , will help you 

Good Luck 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3180379/

http://www.ihcworld.com/_protocols/antibody_protocols/gad67_ihcworld.htm

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0056901

I used separate antibodies to probe for GAD65 and GAD67 (mouse anti-GAD65 (1:10,000 in FIHC; Abcam, ab26113), mouse anti-GAD67 (1:10,000 in FIHC; Chemicon, MAB5406)).  I initially followed the protocol of Hoffman et al., 2008 (http://www.ncbi.nlm.nih.gov/pubmed/18972376) for titrating the antibodies.  

Antibodies react differently if they are perfused with different chemicals (paraformaldehyde fixation results in different staining than picric acid fixation, even with the same antibody, for example). How are you fixing your tissue?  I also used a cryoprotectant solution and had free-floating tissue slices; staining worked better for IHC and ISH when the slices had been allowed to sit in cryoprotectant for at least a week.

I hope this helps.

Hi Amanda,

in our lab, we had good experience with the mouse anti-GAD67 antibody from Millipore (MAB5406) as well, in free-floating slices of rat brains perfused with 4% PFA. Recently, my colleague Giad Ritov used this for double-staining with cFos: https://www.researchgate.net/publication/283347913_A_novel_approach_to_PTSD_modeling_in_rats_reveals_alternating_patterns_of_limbic_activity_in_different_types_of_stress_reaction

Here is the detailed protocol

- wash free-floating slices 3x 5min with PBS

- Block for 15 min (at room temperature, tumbling) with Background sniper (Biocare Medical) or alternatively 1h with serum (e.g. 5-10% donkey serum in PBS with 0.3% Triton X-100)

- incubate over night (at 4 °C, tumbling) with mouse anti-GAD67 (MAB5406) 1:400 in 3% BSA in PBS with 0.3% Triton-X (if you used serum for blocking, you can dilute the AB also in blocking solution)

- wash 3x 5 min with PBS

- incubate 1h (at room temperature, tumbling) with secondary AB: we used Alexa Fluor 488 donkey-anti-mouse 1:1000 in 2% BSA in PBS with 0.3% Triton-X

- wash 3x 5 min with PBS

- mount and cover slip (e.g. with Immunomount)

You might night to titrate the final concentration of the antibody for your own experiments and maybe adjust also serum concentrations for blocking, in case of too much background.

Good luck & best regards

Anne

Article A novel approach to PTSD modeling in rats reveals alternatin...

just a head's up!  We examined GAD in free floating sections and the system that is under investigation matters.  Cortex is easy; hypothalamus is NOT (GAD65 was not as good as 67 anywhere).  That said I also have found that fixation matters.  If using 4% PFA I advise good post fixation 2-4 days or post fixation of the cut sections for at least 24 hr.  The system does much better when well fixed and PFA is not a rapid fixative all by itself.

it easy to show GAD65/67 neuronal bodies in some regions such cerebral cortex and reticular thalamic nucleus, but in other regions  it is much difficult to show GAD65/67-ir neuronal bodies. It will be easy to show the bodies of GAD by  In situ hybridizatioon, which can be combination  with immunohisotchemistry.

For immunostaining GABAergic cell bodies we find it best to use a GAD67-specific antibody rather than those that recognize both GAD65/67. We have tried a few anti-GAD 67 mAbs, and recently compared tanti-GA67 clone 1G10.2 (Millipore clone 1G10.2 #MAB5406) and  anti-GAD67 clone K-87 (Abcam #ab26116) side by side, which I posted in another forum:

"For immunohistochemistry we followed each supplier’s recommendations (e.g., antigen retrieval prior to K-87) and used a 1/4,000 dilution of each antibody on adult mouse brain sections. Both antibodies worked very well for labeling neuronal cell bodies. The K-87 staining extended more into arborizations as well as nerve terminals (e.g., in cerebellar cortex). GAD67 primarily forms homodimers with itself and stays in the cell body, but some GAD67 forms heterodimers with GAD65 and gets transported to synapses (e.g., PMID: 20805323) so such labeling is expected. If you want to visualize those details the K-87 would be preferable. Otherwise, either 1G10.2 or K-87 would be good choices. Both are often used in the literature. For GAD65 labeling, the GAD6 mAb is excellent for immunoblotting and immunohistochemistry and is widely used in the literature."

I hope that is useful.

As others have noted the success of staining for GAD67 cell bodies depends on the region you are staining. For example, it is more difficult to successfully reveal cell bodies in the brainstem and spinal cord than the hippocampus or cortex. Several of the answers above correctly point you to using GAD67 selective antibodies. To enhance staining you could attempt the protocol in this paper:

http://www.ncbi.nlm.nih.gov/pubmed/16255028

A difference here compared to many is that there is no triton in the antibody incubation medium, because they say:

"No permeabilizing agent was utilized, because preliminary studies demonstrated that the presence of permeablizing agents, such as Triton X-100, or cryosectioning reduced labeling of cell bodies"

We have found this approach to work well. However, it does use peroxidase methods so if you wish fluorescence then you may consider a tyramide signalling amplification approach (TSA, kits can be bought from several sources). We have found that using a biotinylated secondary and streptavidin-fluorophore can work too.

You could attempt to detect GABA. This requires high glutaraldehyde fixation, very thin sections, and no detergent permeabilisation. If you want double staining though, many other antigens become unrecognisable with high glutaraldehyde,

Finally, there are several TG mice with GAD67 cells labelled with GFP. We have found that the knock-in described in paper below is fairly faithful in labelling GAD67 cells in the spinal cord and brainstem and has been used in several other CNS regions:

http://www.ncbi.nlm.nih.gov/pubmed/14574680

Yuchio Yanagawa is the contact for this mouse.

Good luck!