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Please help.

I know how to isolate the different cells (astrocytes, other glial cells, neurons and synaptosomes) from brain tissue using a Ficoll-based separation but how do I determine the purity of the fractions that I have isolated if I cannot use immunological techniques, FACS or SEM analysis.

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You can do qPCR for certain markers from the extract. GFAP for astrocytes, Tuj1 for neurons, Myelin for oligodendrocytes etc and calculate their percentages to have an estimate of the relative populations. If you are quite used to microscopy and studying these cells then you can identify the different populations of cells with normal light microscopy as well; this is not quantitative and is susceptible to bias.

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  • $\begingroup$ q-PCR would be the most accurate PCR technique if you wanted a breakdown of the population by percentage. Simple optical techniques are quantitative if you abide by stereology techniques. $\endgroup$
    – user560
    Commented Sep 20, 2014 at 11:55
  • $\begingroup$ I guess that a Western would count as an example of an immunological technique (excluded in the question). $\endgroup$
    – Alan Boyd
    Commented Sep 20, 2014 at 15:41
  • $\begingroup$ yeah I meant qPCR only.. @AlanBoyd.. yeah true 'ill correct it $\endgroup$
    – WYSIWYG
    Commented Sep 21, 2014 at 5:26
  • $\begingroup$ @leonardo.. they are quantitative if there is an automated algorithm for scanning and classification of cells based on the images. $\endgroup$
    – WYSIWYG
    Commented Sep 21, 2014 at 5:50
  • $\begingroup$ @WYSIWYG - I agree, but that means the technique is not only qualitative. Automated imaging and segmentation is the modern successor to random fields + stereology. $\endgroup$
    – user560
    Commented Sep 21, 2014 at 19:35

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