I have cultures of astrocytes (derived from E15 wistar rats) and I'm trying to characterize the culture, but after ICQ just few of them express GFAP and the others doesn´t expres or the expression is to low? What can I do? increase Antibody dilution? change antybody?
This is a quote from a review by Sofroniew and Vinters 2010 Acta Neuropthol 119:7-35.
Immunohistochemical techniques that enable the detection of specific molecular markers at the single-cell level are essential tools for identifying and characterizing cells in healthy and pathological tissue. Expression of glial fibrillary acid protein (GFAP) has become a prototypical marker for immunohistochemical identification of astrocytes. Nevertheless, it is important to recognize the appropriate uses and limitations of GFAP as an astrocyte marker. GFAP was first isolated as a protein highly concentrated in old demyelinated plaques from multiple sclerosis patients and was then found to be associated immunohistochemically with reactive astrocytes in such plaques and in other pathological contexts [62, 63]. In line with this original mode of identification, GFAP expression can be regarded as a sensitive
and reliable marker that labels most, if not all, reactive astrocytes that are responding to CNS injuries (Fig. 3). However, GFAP is not an absolute marker of all non-reactive astrocytes and is often not immunohistochemically detectable in astrocytes in healthy CNS tissue or remote from CNS lesions (Figs. 3a, 4a). Although GFAP is immunohistochemically detectable in many astrocytes throughout the healthy CNS, it is clear from double staining with multiple markers (including transgenic reporter proteins) that many mature astrocytes in healthy CNS tissue do not express detectable levels of GFAP and that GFAP expression by astrocytes exhibits both regional and local variability that is dynamically regulated by a large number of inter- and intra-cellular signaling molecules [226].
You can try antibody to S100B. We have had success with this marker in tissue, but we haven't tried it with cultured astrocytes.
Dear James, Thank you very much now it makes sense, You were a great help.
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