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different staining intensity


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#1 Minnie Mouse

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Posted 15 April 2009 - 08:17 PM

Why is the staining intensity weaker in fixed permeabilized cells than non-fixed cells?

1. The permeabilized cancer cells were fixed with 3.7% paraformaldehyde and then permeabilized with saponin.
2. cells were stained with either A or B antibody
3. the secondary conjugate was anti-mouse FITC


The antigen was expressed on the cell surface.
Two monoclonal antibodies (A and B ) against the same antigen were used to stain the cells.
A and B antibodies stained weaker in fixed permeabilized cells than non-fixed cells.

Attached File  Minnie__s_flow_cytometry_result.ppt   56KB   208 downloads


Why?



Thanks in advance.

#2 Thapa

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Posted 15 April 2009 - 10:31 PM

I dont know the exact reason why. But I have read similar information when I used monoclonal antibody ICAM-1 (R&D). Although I didnt perform/check the suitability, what they have written in product sheet that "this antibody is not suitable for labelling formalin fixed tissue". So, I did just another attempt with methanol fixation....just blindly following previous published article.

May be the same in your case, your mAbs are not suitable under PFA fixation.
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#3 almost a doctor

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Posted 15 April 2009 - 11:47 PM

Why is the staining intensity weaker in fixed permeabilized cells than non-fixed cells?

1. The permeabilized cancer cells were fixed with 3.7% paraformaldehyde and then permeabilized with saponin.
2. cells were stained with either A or B antibody
3. the secondary conjugate was anti-mouse FITC


The antigen was expressed on the cell surface.
Two monoclonal antibodies (A and B ) against the same antigen were used to stain the cells.
A and B antibodies stained weaker in fixed permeabilized cells than non-fixed cells.

Attached File  Minnie__s_flow_cytometry_result.ppt   56KB   208 downloads


Why?



Thanks in advance.


From your post I understand that you are staining after fixation and permeabilization. If so, the treatment (mos probably PFA but maybe saponin too) is changing the distribution of your surface antigen. Incubate your cells with A and/or B then fix and permeabilize and have a look at the staining, this should hopefully look more similar to non-fixed cells.

Why are you fixing and permeabilizing if you are interested in surface antigens? I asume you want to do intracellular staining after A and B. If so, my advice is this: stain the extracellular markers first A/B, followed by 2nd antibody, then fix, then permeabilize and then stain for the intracellular markers.

Good luck! :(




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