I am looking for a good antibody to label hilar interneurons in the hippocampus. I have tried several antibodies against GABA and GAD from millipore but did not get good signals. Can someone recommend some antibodies that work well? Thank you.
One question would be what is the antibody isoform and also the protocol you are using for GAD? Are you using the GAD65/67 isoform from Millipore, this generally gives a more robust labeling profile when used at 1:1000-2000. We use triton-X100 at 0.3-0.5% to aid permeability. As said above specific interneuron markers would be a good alternative step. As Just Genius stated, he PV, CB and CR from SWANT (1:5000, 1:2000, 1:2000, respectively) are excellent antibodies, and probably some of the best on the market. The somatostatin from Bachem (1:2000) is an excellent marker for HIPP type cells. You could also consider using a CCK antibody which labels HICAP cells, we have used the pre/pro-CCK from Masahiko Watanabe, which gave excellent results.
hope this helps
Hi! Have you also considered such popular interneuron markers, like calretinin, calbindin and parvalbumin? Interneurons expressing these markers may well be found in the hilar region of the hippocampus (but please check in literature before trying). Years ago I have worked with antibodies from Swant/Bellinzona for IHC in rat brains and was quite satisfied. Singular neurons positive for PAV or calretinin were found in the hilar region.
Good luck!
If you are looking for hilar interneurons, some of them express somatostatin, substance P apart from above mentioned neuronal markers.
Good Luck.
One question would be what is the antibody isoform and also the protocol you are using for GAD? Are you using the GAD65/67 isoform from Millipore, this generally gives a more robust labeling profile when used at 1:1000-2000. We use triton-X100 at 0.3-0.5% to aid permeability. As said above specific interneuron markers would be a good alternative step. As Just Genius stated, he PV, CB and CR from SWANT (1:5000, 1:2000, 1:2000, respectively) are excellent antibodies, and probably some of the best on the market. The somatostatin from Bachem (1:2000) is an excellent marker for HIPP type cells. You could also consider using a CCK antibody which labels HICAP cells, we have used the pre/pro-CCK from Masahiko Watanabe, which gave excellent results.
hope this helps
I use GAD67 Millipore (1:500) after permeabilizing with tritron 0.3% I use 10% normal goat serum as blocking agent for 30 min and it works perfectly.
Antibodies for calretinin, calbindin and parvalbumin from Swant/Bellinzona worked really well for me too. You can also try SIGMA P3088 for parvalbumin or GAD-65 from Novus (NB110-41528) or GAD-67 form Millipore (MAB5406)
Good luck!
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