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antigen detection in immunocytochemistry


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#1 dnasequencer

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Posted 24 November 2014 - 11:09 AM

Can an antibody bind to an antigen present inside a vesicle in the cell, in immunocytochemistry?



#2 bob1

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Posted 24 November 2014 - 11:37 AM

Yes - given sufficient permeabilization of the vesicle.



#3 dnasequencer

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Posted 24 November 2014 - 10:40 PM

Thank you bob 1, but can an Ig detect this antigen without premeabilization?

In my experiment I got voltage-gated sodium channel protein in the cytoplasm, so I though it might represent the proteins in vesicles transporting from Golgi to the plasma membrane, but I just fixed my cells, did not premeabilize them! 



#4 bob1

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Posted 24 November 2014 - 11:09 PM

Generally no, IgG won't enter the cell without permeabilizatio, as it is too large. However, if you used a methanol/acetone fix you probably have enough holes in the cell for it to work. Otherwise incubating for 10 min in 0.01% Triton X100 in TBS or PBS will work.



#5 dnasequencer

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Posted 25 November 2014 - 02:15 AM

Thanks bob

Now it makes scene, I fixed my cells which 3.7% formaldehyde which I prepared from a 37% stock formaldehyde solution, this solution contains 10-15% methanol as a stabilizer. So my cells probably got premeabilized by the methanol available in the formaldehyde. 



#6 bob1

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Posted 25 November 2014 - 09:44 AM

Hmmm, that's not normally enough to do a proper permeabilization. How did your controls look? Were you using an antibody that other people have used successfully for ICC?



#7 dnasequencer

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Posted 25 November 2014 - 09:50 AM

I have got no signals in the negative controls and yes this antibody was used before in the literature and got same results as mine.

But how do I explain the presence of a protein, that is normally expressed in the plasma membrane, in the cytoplasm?



#8 bob1

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Posted 25 November 2014 - 11:48 AM

Are you using confocal microscopy to see the staining pattern, or just a conventional epifluorescence microscope?

 

If the latter - think about the cell as a 3D shape, and consider where the membrane is relative to the 2D picture you are taking...

 

Another thing to consider is that the antibody may have some non-specific binding. Is it possible to get a Na+ channel negative (i.e. -/-) cell to test this on? If you are running westerns and get a single band with the antibody (i.e. no other bands present), and are using the antibody at the same concentration for ICC, then you can be reasonably confident of it being specific staining.






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