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Eliminating non-specific staining for ICC
Posted by: troubled PhD student (IP Hidden, New member, 2)
Date: August 15, 2005 05:56AM

Hi there, I'm new to this forum and am really hoping that someone can help me with my problem of non-specific staining. I am using brain tumour cells and looking for NG2. However, all the negative control coverslips keep showing the same staining pattern as the 'test' ones. I have tried to eliminate this staining by incubating with serum raised to the secondary antibody, raising the NaCl to 2.5% in the rinising buffer, raising the pH to 9 in the rinsing buffer, adding 0.05% tween 20 to buffer, but to no avail! I really do not know what to try next. Any suggestions would be greatly appreciated.

 

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Re: Eliminating non-specific staining for ICC
Posted by: mito lab (IP Hidden, Unregistered user, )
Date: August 15, 2005 09:57PM

you need to clarify little bit your staining condition:

1. species of the brain tumor cell lines originate, mouse? human?
2. primary antibody NG2: mouse monoclonal? rabbit polyclonal?
3. negative control: no primary antibody control or negative NG2 expression cell line control?
4. concentration of primary and secondary antibody?
5. permeabilization condition? staining condition? washing condition?

 

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Re: Eliminating non-specific staining for ICC
Posted by: troubled PhD student (IP Hidden, New member, 2)
Date: August 17, 2005 05:29AM

Hi, sorry for not clarifying things! In response to your questions
1. Cell lines are of human origin
2. I have used both mouse and rabbit primary antibodies with their respective secondaries
3. The negative control I use is omitting the primary antibody
4. primary rabbit 1:500, secondary anti-rabbit 1:100, primary mouse 1:200, secondary anti-rabbit 1:100
5. permeablize with 0.2% triton x-100 and wash with PBS (with the additions I mentioned on the previous mail

Thanks again, hope this makes things a little clearer.

 

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Re: Eliminating non-specific staining for ICC
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: August 19, 2005 09:11AM

Maybe you can try more diluted secondary antibody and shorten the incubation time. Using normal rabbit serum may also be helpful. Good luck!

 

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