Hey there,
I'm looking for a selective astrocyte marker which does not stains other glial cells, especially schwann cells. I usally use GFAP but I found at a paper, that it also labels schwann cells and a minor subpopulation of oligodendrocytes in SVZ. The same goes for the S100-Protein. I also looked up MANF (Mesencephalic Astrocyte-Derived Neurotrophic Factor) but it seems to be located to the ventral mid-brain and the substantia nigra. The astrocytes I like to stain come from the cochlear nucleus (brain stem).
Any ideas?
Christina:
A good option might be GLAST (sometimes called EAAT). This gets all astrocytes + radial astrocytes, without (to my knowledge) getting other glial cells. However, I don't know its specificity in the CN.
Hey Pete,
I looked it up. GLAST ist also expressed in schwann cell myelin, neurons and satellite cells of spiral ganglion and additionally oligodendrocytes.
Thanks for the hint.
http://citeseerx.ist.psu.edu/viewdoc/download;jsessionid=AC57526FE7CB231DEA3EB1F6A30A6687?doi=10.1.1.377.5713&rep=rep1&type=pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2667548/
Hi Christina,
What about vimentin. I know that it primarily labels radial glia and astrocytes. You may also consider targeting more than one marker. Perhaps using couple of markers, GFAP and vimentin together and GFAP and GLAST or EAAT2 together may help you. In the past I have found that TASK-1 and NHERF molecules also localized to the astrocytes, but I am noy sure how specific is that.
best wishes,
Refik
Hey Rifik,
thanks for your suggestions. I found that vimentin is expressed in the cytoskeletion of SC and neurons and TASK-1 is also expressed in SC but only in carotid bodies. So I will try to exclude astrocytes by a triple staining with S100, GFAP and MPZ. I hope that it may works.
best regards,
Christina
http://dev.biologists.org/content/139/7/1359.long
Article TASK-1, TASK-2, TASK-3 and TRAAK immunoreactivities in the r...
Hi Christina,
Sounds great and sounds like you know pretty much what you are. Cochlear nucleus is special to study with interesting its synaptic organization. Thank you for sharing it with us. I have previously used astrocyte labelling, but never had to do just astrocyte labelling. Looks like multiple labelling is currently one of your options. If I hear anythingelse I will let you know.
All the best,
Refik
Thank you, Rafik.
I will let you know, if the triple staining works.
Best regards,
Christina
I have been using glutamine synthetase antibody which in cortex gives nice cytosolic astrocyte labelling. No neurons stained for sure. Not sure about other glial populations, but this paper suggests no strong oligodendrocyte labelling:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3797418/
Hi Roger,
I already looked GS up. It's highly expressed in differentiated SC.
But thanks for trying.
Best regards,
Christina
http://www.ncbi.nlm.nih.gov/pubmed/20107048
Hi Christina,
I've heard good things about Aldh1L1. It's supposed to be a highly specific astrocyte marker in the brain, albeit somewhat challenging to stain for.
Best wishes,
Marcin
Hey Marcin,
I'm sorry to say that Aldh1L1 is also a SC marker.
Thanks for trying.
Best regards,
Christina
http://jnci.oxfordjournals.org/content/106/8/dju184.full
Oh well, you got me :) Thanks for letting me know and good luck with the triple staining!
Staining is done. It is not one of the best stainings but sufficient. I found single positive, double positive or triple positive cells. So there are Schwann cells (MPZ+/S100+/GFAP+; MPZ+/S100+; MPZ+/GFAP+) and also cells which are GFAP+/S100+. These cells are may be Astrocytes or Oligodendrocytes.
NDRG2 is a new marker for astrocytic processes and cells (Flugge et al, 2014, Cell Tissue Res), but I have not worked with this again. Maybe you can try it.
You can try with Glutamine synthetase, an enzyme in astrocytes to convert glutamate to glutamine.
Hi Patro,
as I already mentioned GS is highly expressed in differentiation SC. But thanks for trying.
Dear Christina, indeed, we used several specific astrocyte markers (GS, GLAST, GLT-1, OCT-1, OCT-2, OCT-3, Kir4.1, Kir6.1,etc, however the staining is strongly dependent on age. For example, GLT-1, OCT-3, Kir4.1 and Kir6.1 are stably expressed in rat and mouse after 11-21 days, except if cells are transormed to cancer cells as glioblastoma.
If you are looking for healthy adults, try SLC22A-3 (organic cation transporter, OCT-3) or SLC18B1 (vesicular polyamine transporter, vPAt), or directly try spermine and spermidine. Check it. The markers would be very interesting for you. I can provide more information if need.
Cordially, Serguei.
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