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Immunofluorescence bad signal


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4 replies to this topic

#1 alessandro

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Posted 01 February 2010 - 11:16 AM

Hi to all, i have to ask this question... I recently made an immunofluorescence of cells fixed with paraphormaldeyde 3,7% 3weeks ago...and the signal of my two phluorophore is very low, or totally absent... I wonder if this problem is dued to the many days the cells were kept in fixing solution...

#2 bob1

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Posted 01 February 2010 - 03:14 PM

It could well be too long a storage time... the usual guide is to only keep PFA fixed cells for a week before the fixation starts to degrade. For longer term storage dehydrate in ethanol or methanol.

#3 NemaToStella

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Posted 17 February 2010 - 02:59 PM

With fixing solution, do you actually mean the buffer you fixed the cells in (without PFA) or buffer + PFA?

#4 bob1

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Posted 17 February 2010 - 03:14 PM

Just buffer, if you keep them in the PFA for too long it will "over fix" the cells which will mean that you will have difficulty getting the epitopes exposed for antibody binding. PFA will also degrade after about a week in the fridge.

#5 NemaToStella

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Posted 18 February 2010 - 01:11 PM

Yep, that's why I was asking :P, and that could be the problem.




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