Hi, Staining for Glycine itself is usually a pretty good marker for glycinergic cells. The intracellular glycine content is usually much higher in glycinergic cells than in other cells, although one needs to be careful with that. Glycine labeling could definitely be worthwhile. Good anti-glycine antibodies are available from several companies Signature Immunologics makes a very good one and Chemicon also has a very good one (now sold by Millipore). (In retina for instance there are many glycinergic amacrine cells that stain beautifully for glycine, but a subpopulation of bipolar cells, that are not glycinergic also show disctinct labeling due to diffusion of glycine from amacrine cells into bipolar cells via gap junctions). The other amino acid transmitters also can be immunolabeled very effectively (GABA, Glutamate, etc). If you decide to pursue this, look carefully at the fixation requirements of the antibodies- some require some amount of glutaraldehyde in the fixative, some do not.  An added point on glycine and amino acid labeling is that it works beautifully in plastic sections and is compatible with post-embedding labeling at the light and EM levels.

    Plasma membrane Glycine transporters are also excellent markers for glycinergic neurons. GlyT2 is typically the transporter used by neurons. There are GlyT2 antibodies available, but I have not worked specifically with any of them myself. Millipore sells one that works for immunolabeling (they show a picture of it on the website) and so does Synaptic Systems (I have had very good luck with Synaptic Systems antibodies, in general). Abcam also has one.  GlyT1 is typically the transporter used by glial cells (although there are exceptions, like retinal amacrine cells). There is a very good GlyT1 antibody available from Millipore.

   If there is not enough pain in your life already, you could try Glycine uptake autoradiography. It works, but in order to visualize the neurons specifically it would probably be necessary to block glial uptake.

    The vesicular transporter for glycine on synaptic vesicles would not be a good marker, as the same transporter is also used for loading GABA into vesicles in GABAergic cells.

   Good luck.

Dave Sherry

I will try it. For the GlyT2 I think we have already tried it and it didnt work but I will give it a shot. Thank you very much for your reply, it helped me a lot.

Best,

Evangelia Fourli

Hi Evangelia,

I'm working for Synaptic Systems. I can offer you a free sample of one of our anti-GlyT2 abs. Both have been tested in mouse spinal cord (please have a look at the images);

https://www.sysy.com/products/glyctrans/index.php

If you are interested, please contact me at [email protected]

Best regards,

Carsten