We usually use anti-GFAP to label astrocytes, but this usually labels only reactive astrocytes. Has anyone used anything different? We are trying to see if a certain metabolic enzyme is expressed by both neurons and astrocytes- so we don't want to limit ourselves to only reactive glia.
In my experience, GFAP labels most astrocytes not solely reactive ones. GFAP can be used to assay for reactive glia, but in and of itself is not a marker for reactive glia. GFAP does have some issues with staining in the cortex though. But it is definitely not solely reactive astrocytes, and were that the case, then I have some problems with my data as in a WT mouse with no manipulations done to it the majority of the brain has gone reactive :-)
There are several antibodies that you could use to get what the majority of the field will call widespread astrocyte marker that can be used instead of GFAP.
Glutamine synthetase (GS) - metabolic enzyme in astrocytes which will give cytosolic labeling
Glt-1 - glutamate transporter primarily expressed by astrocytes
GLAST - glutamate transporter primarily expressed by astrocytes
Aldh1L1 - supposed to be one of the best astrocyte specific proteins, but tends to be difficult to stain for.
S100-beta: another fairly astrocyte specific cytosolic protein.
Also, you may want to consider using an astrocyte specific reporter mouse. There is a GFAP::EGFP mouse, Glt-1::EGFP, and Aldh1L1::EGFP mice available from both Jackson Labs and GenSat.
In my experience, GFAP labels most astrocytes not solely reactive ones. GFAP can be used to assay for reactive glia, but in and of itself is not a marker for reactive glia. GFAP does have some issues with staining in the cortex though. But it is definitely not solely reactive astrocytes, and were that the case, then I have some problems with my data as in a WT mouse with no manipulations done to it the majority of the brain has gone reactive :-)
There are several antibodies that you could use to get what the majority of the field will call widespread astrocyte marker that can be used instead of GFAP.
Glutamine synthetase (GS) - metabolic enzyme in astrocytes which will give cytosolic labeling
Glt-1 - glutamate transporter primarily expressed by astrocytes
GLAST - glutamate transporter primarily expressed by astrocytes
Aldh1L1 - supposed to be one of the best astrocyte specific proteins, but tends to be difficult to stain for.
S100-beta: another fairly astrocyte specific cytosolic protein.
Also, you may want to consider using an astrocyte specific reporter mouse. There is a GFAP::EGFP mouse, Glt-1::EGFP, and Aldh1L1::EGFP mice available from both Jackson Labs and GenSat.
You've gotten some good recommendations. Just wanted to echo the previous statement - I dont think there is a way to label all astrocytes in the brain. This is becoming an increasingly important point in that we need to give up the idea of astrocytes as one homogenous population and realize that there are, and more will be elucidated, many functional and structural differences in astrocytes and very likely in different across different species.
Thanks everyone for all the answers and advice! I love that we can have this type of interaction.
Jeremy has replied you beautifully. I would recommend you three practical markers for astrocytes according to my experience;
Glt-1, S-100-beta and G-FAP
I think these are sufficient
to my experience, if some human astrocytes have barely detectable GFAP, grey matter ans white matter astrocytes in mouse CNS are stained well with many GFAP antibodies . The staining is easier to detect on reactive astrocyte.
Anyone considered nuclear markers?
I think Olig2 might work, but I don't have any personal experience using it.
Olig2 stains oligodendrocyte lineage cells, not astrocytes. I found the Aldh1l1-eGFP mouse nicely labeling all astroyte cell bodies. Aldh1l1 staining works, but is not that great. I am not aware of an astrocyte-specific nuclear marker. In my experience GFAP does not label most of the astrocytes in the intact cortical gray matter in mice.
AlphaB crystallin (cryab gene) is expressed in astrocytes and oligodendrocytes in brain and in cultured cells, please see the article in the link below.
http://www.ncbi.nlm.nih.gov/pubmed/15339919
RKG
As most of the responders suggesting, anti Glial Fibrillary Acidic Protein (GFAP) should work for both protoplasmic and Fibrous Astrocytes. Anti S-100 (Calcium binding protein) and Anti Glutamine synthetase should also work. These both have been told to be marker for astrocytes (Reference: Basic neurochemistry, Seagel et al.) but I am really not sure about later two. I think you should go with GFAP.
Our experience that GFAP should stain all astrocyte not only reactive astrocyte. however if you are not confirm then you can use astrocyte specific other marker like connexin 43.
Ben Barres had a paper a while back (I think J. Neurosci) that showed Aldh1l1 being more inclusive of astrocytes than GFAP. I forget whether he showed that GFAP stained for predominantly grey or white matter astrocytes.
Like other people stated, there is an Aldh1l1-GFP mouse. I remember people in David Rowitch's lab at UCSF using that mouse, and claim to see more GFP expression in their mouse brains compared to the GFAP-GFP reporter line.
I've tried the antibody before, and I think it was terrible. That's how I found out about the Rowitch lab next door using the mouse reporter line b/c you can actually see flourescent cells w/ them.
Just to reiterate what others have already said. GFAP is a good general marker, but does tend to mark a subset of astrocytes. Aldh1L1 will give a better picture if you can get a good antibody, as GFAP doesn't always label all the processes of the astrocyte as well. Hope this helps, and great discussion!
Would it be valid to identify astrocytes in cell culture using sulforhodamine 101?
I have no other astrocyte marker that GFAP, but I am being required for an additional one because "GFAP is a promiscuous antibody". However, I have SR101 and I'd like to know if that would be an acceptable control to identify astrocytes in cell culture.
Thank you all for a very informative and interesting discussion. I am looking for a cell filling astrocyte marker and it seems like Glutamine synthetase could be very promising! Which antibody works best in your hand for immunofluorescence on mouse brain slices (PFA fixed, vibratome sliced)? Have any of you used it for labeling astrocyte in the central nervous system? I found a few articles successfully used it for the retina but haven't seen much in cortex or thalamus. Thank so much!!! Best, Huong
Dear Marijke De bock,
we use an antibody against glutamine synthethase, a key enzyme in astrocytes which works beautifzully see Rollenhagen et al., Brain Structure and Function 2014. If you have any further questions please do not hesitate to contact me.
Best wishes
Joe Lübke
Aldh1L1 is a highly specific antigenic marker for astrocytes with a substantially broader pattern of astrocyte expression than the traditional astrocyte marker GFAP.
Hi Everyone,
Quite new in the field of astrocytes but I have heard that GFAP doesn't seem to be the best marker for astrocytes anymore? Due to the fact that it labels mostly glial cells in general including olfactory ensheathing cells and microglia sometimes... but also neuroblast...If you really want to label only astrocytes what you would recommend? S100Beta or one of the Glutamate transporter? What the Aldh1L1 is targeting in the astrocyte?
Thanks in advance
Hi Kimberly B Bjugstad and all,
After 6 years of your question, we still do not have a marker for all astrocytes, and because of the enormous heterogeneity of these cells, I am not optimistic about the existence of a universal marker to label all astroglial leneage cells. Although there are many good markers, as answerd above, your choice should be based on brain`s area/circuit and astrocyte population of interest.
Bests,
Marcus
I'm looking for an astrocytes marker in the mouse nucleus accumbens other than GFAP. Thanks
S100b can be a backup marker as well but GFAP works fine, you would also want to be careful to use 1 marker since GFAP is also expressed by cells during neurogenesis. To further delineate the between astrocytes and neurons you can use UCHL1 and NeuN for neurons but essentially there is no one global marker for any cell type in the end.
What about Vimentin? and has anyone have problem with western in S100beta?
- Badges
- Science method