Today, we processed tissue from our first GFAP-EGFP mouse (on a C57B6 background). Tissue was fixed with 4% PFA and imaged using a 1.4NA 63x oil immersion lens. We compared this side by side with a section taken from a C57B6 mouse processed using a commercially available antibody from Millipore - also fixed with 4% PFA. The immunohistochemically identified GFAP - is really good but principally only reveals primary and secondary and occasionally tertiary branching. The labelling clearly reveals the major features of the cytoskeleton but nothing more. In contrast the GFAP-EGFP labelling was incredibly intense - and the primary structure was difficult to visualise for the thousands of tiny hairlike branches that made the astrocyte appear to be a fluffy little ball. The differences between the two approaches yield stark differences. What is going on here and what should I believe? Is the labelling from the GFAP-EGFP mice real? Is this really representing what the GFAP cytoskeleton really looks like? Or is this a quirk of genetic wizardry - and the real one to believe is the immunolabelling.
Hi Rohan, in my opinion you see different things. With the immunohistochemistry against GFAP you are revealing the cytoskeleton, but in the transgenic mice GFAP-EGFP you induce the synthesis of the green protein under GFAP promotor, but the green protein will diffuse throughout the cytoplasm of all extensions, even the little ones where there is not GFAP.
I think you're probably seeing is the actual morphology of an astrocyte and no the "skeleton". Probably the images you are seeing are very close to the images of astrocytes with Golgi staining
Will be nice to see a picture!
Cheers
Bernardo
Hi Rohan, in my opinion you see different things. With the immunohistochemistry against GFAP you are revealing the cytoskeleton, but in the transgenic mice GFAP-EGFP you induce the synthesis of the green protein under GFAP promotor, but the green protein will diffuse throughout the cytoplasm of all extensions, even the little ones where there is not GFAP.
I think you're probably seeing is the actual morphology of an astrocyte and no the "skeleton". Probably the images you are seeing are very close to the images of astrocytes with Golgi staining
Will be nice to see a picture!
Cheers
Bernardo
Thanks Bernardo - I really like that explanation. I will post a couple of images later today for illustration. Certainly, the GFAP-EGFP looks exactly like a Golgi/Iontophoretically dye filled astrocyte.
Bernardo is correct. The antibody staining only lables the location of the GFAP protein within the cells, which makes up the primary processes of the cytoskeleton. The trangenic cells express GFP using the GFAP promoter to drive expression, hence only astrocytes will have the reporter. You will never get the full morphology of an astrocyte using IHC against GFAP, you wont even see the soma clearly. There is no genetic wizarding at work, just different techiniques for labeling cells.
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