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Immunocytochemistry neurons problems with Triton X 100


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#1 Martina Bigoli

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Posted 10 February 2015 - 01:23 PM

good evening,

 

I am working on neurons. Today I was wrong with the dilution of Triton X100 to permeabilize the cells. I should prepare 0.1% Triton X 100 but I realized that I prepared 0.8% Triton X 100. My staining is on MAP2 and on synaptic markers (synaptophisin, PSD-95, VGLUT1, VGAT, Gephyrin)

 

How much this mistake affects the experiment?

Thanks.



#2 bob1

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Posted 10 February 2015 - 03:51 PM

Depends on the antibody and where it is in the cell, fixation method, and subsequent steps. TritonX100 is quite strong and is used to make holes in the cellular membranes, too much of it could damage the cell so that proteins are no longer held tightly in a cross-linked form... still the only way to know is to look and see what happened, but having said that, you will still have to repeat the experiment to make sure that it didn't alter the staining you see.






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