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Staining Neurons and counting them using CLARITY?

maryam_zmaryam_z Posts: 2
in Sample Staining
we are using the CLARITY technique to clear Rat brains and we would like to label astrocytes and neurons in the cerebral cortex and to be able to count them. We tried to stain it using NeuN and GFAP, but it appears that it didn’t stain when we did imaging. Do you have any recommendations/thoughts on why it did not work, or any specific steps we should follow?
we followed steps on
http://wiki.claritytechniques.org/index.php/Immunostaining
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Comments

  • aysegulaysegul Posts: 33
    Hi maryam_z, how many days did you incubate your section in primer antibody and how much antibody dilution did you use?
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  • maryam_zmaryam_z Posts: 2
    Hello aysegul. We incubated a 200 micrometer slides for 1 day and 2 days in 1:50 concentration primary antibody. We incubated them in 2 different temp: one at 37 degrees, and the other at 4 degrees. The same time and temp was repeated for secondaries antibodies.
    GFAP did stain, but NeuN didn't in any of the scenarios.
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  • xyangxyang Posts: 5
    Hi maryam_z,
    I am doing the same staining of NeuN and GFAP on mouse brain slice (1 mm) and am having the same issue. GFAP did get stained, but NeuN didn't. For me I guess the issue comes from leaving the sample in blocking solution for too long (37C 24 hr). Did you block the sample?
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  • MariaSuessmilchMariaSuessmilch Posts: 5
    Did blocking made a difference for you? I don't have this step in my original protocol...

    Also, can I ask which antibodies exactly you used? So which company it's from and what product code it has?
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