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Author Topic: Methods for Immuno staining using live (unfixed) cells  (Read 6792 times)

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Offline awlifland

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Methods for Immuno staining using live (unfixed) cells
« on: October 22, 2007, 02:00:34 PM »
Hi Everyone,

I'm looking for protocols for immunolabeling surface cell markers on unfixed cells. It is important for the staining (Ab recognition) to be done on unfixed as the goal of the project will involve live animal experiments (in which the cells obviously cannot be fixed), however, after the antibodies are bound I will fix them for microscopy. So far I have done a standard IIF staining protocol except that I fix after the secondary is bound (incubate with primary, wash, secondary, wash, fix. all at 4degC). I have a fairly low signal to noise and any suggestions to improve this would be greatly appreciated.

Thanks
-Aaron

Methods for Immuno staining using live (unfixed) cells
« on: October 22, 2007, 02:00:34 PM »

Offline richard03

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Re: Methods for Immuno staining using live (unfixed) cells
« Reply #1 on: October 22, 2007, 07:08:20 PM »
What tissue cells do you study? Are the cells cultured cells?

Offline awlifland

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Re: Methods for Immuno staining using live (unfixed) cells
« Reply #2 on: October 22, 2007, 09:14:08 PM »
They're veros.

Offline richard03

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Re: Methods for Immuno staining using live (unfixed) cells
« Reply #3 on: October 22, 2007, 10:12:51 PM »
Can you fix the cells after primary antibody incubation? It may perform better than fixing after secondary antibody. Also you may need more antibodies/higher concentration for live cell binding than fixed cell binding.

Re: Methods for Immuno staining using live (unfixed) cells
« Reply #3 on: October 22, 2007, 10:12:51 PM »