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Author Topic: Anti-cMyc on brain tissue  (Read 2400 times)

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Offline NKakava

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Anti-cMyc on brain tissue
« on: February 24, 2014, 04:04:51 PM »
Hello all,

I will use the anti-cmyc antibody on adult rat perfused brain tissue (40 um thick). The antibody has been tested in my lab already but I have a concern about my staining. I am expecting the myc epitope to be localized in the nucleus. That is because i have cloned a nuclear targeting sequence, which targets the myc into the nucleus, into a AAV virus and injected rats. My concern is about how I will do the permeabilization. Do you think that Triton (in a concentration of 0,25% or 0,5%) will be enough to permeabilize the nuclear membrane when blocking? Or should I do an antigen retrieval? Any suggestions will be useful.

That's the current protocol I am testing:

3x10' washes in PBS
1 hr blocking (5% goat serum, 0,25 or 0,5% triton X-100 in PBS)
3x5' washes in PBS
O/N incubation with mouse anti-cmyc (1:250, 1:500, 1:1000) in 2% goat serum, 0,25% triton, PBS
3x5' washes in PBS
1 hr incubation with goat anti-mouse (1:500) in 2% goat serum, 0,25% triton, PBS
3x10' washes in PBS

Anti-cMyc on brain tissue
« on: February 24, 2014, 04:04:51 PM »