I have attempted staining primary cortical neurons from mouse in order to identify the percentage of astrocytes.
I used GFAP rabbit (Z0334) from Dako and NeuN mouse (MAB377/X) from Milipore, both at a dilution of 1:500 in a blocking solution consisting of 10% goat serum in 1%BSA/ 0.1% Triton X/ 1x PBS.
For some reason, while neurons are stained successfully, GFAP overlaps the neurons instead of marking the astrocytes.
Can anyone provide any insight on what could be the problem? My colleagues have tried the GFAP antibody on sections and it always works. Is the concentration of Triton X maybe too high?
Thank you all so much for taking the time to help!
how long your cultured neurons in vitro when you used for staining?
It seems your NeuN staining was too strong, the neuron process was stained. The green fluorescence may be due to strong NeuN staining, it is not specific staining. My suggestion is increasing your NeuN antibody (1:1000-2000) and secondary antibody dilution (1:1000-5000).
Hi!
Thanks so much for the quick response! The neurons were 8 days in vitro when I fixed them with PFA to do the staining. I will definitely try increasing the dilution as you suggested! My colleagues use it at about 1:800. I can try with 1:1000 while keeping GFAP at 1:500! Thanks!
If you have enough samples, you can try to stain with GFAP and NeuN respectively. It will help you figure out whether the stain is specific and didn't affect by another fluorescence signal .
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