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inmunofluorescence artifacts


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#1 sela

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Posted 03 March 2009 - 10:20 AM

Hi all, :)
Does anybody know if fixing and permeabilizing with methanol (with or without PF) generates artifacts? and the use of different serums to block?

#2 madscientist750

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Posted 04 March 2009 - 07:58 AM

Hi,

Glad to see someone else interested in fluorescence work on this forum. Based on my work I have always found working with methanol to tricky. What kind of staining are you trying to accomplish? Often I am able side step the issues of methanol with: gluderaldyhide fixation, 1% BSA in PBS incubation, then acetone permiblization. I understand that my "work around" does not work under all conditions, but it might be woth a try.

#3 pmaj

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Posted 06 March 2009 - 07:50 AM

Hi all, :)
Does anybody know if fixing and permeabilizing with methanol (with or without PF) generates artifacts? and the use of different serums to block?


Hi there,

I have used 4% formaldehyde prepared in PBS and it seems to work fine. I fix the cells with 4% formaldehyde for 8-10 min on ice and then continue further with the protocol.

Hope this helps

Pmaj
There is a fine line between persistence and obstinacy. I have come to realize the key is to choose a problem that is worth persistent effort.
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#4 WOW

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Posted 15 March 2009 - 08:22 PM

Hi,

Glad to see someone else interested in fluorescence work on this forum. Based on my work I have always found working with methanol to tricky. What kind of staining are you trying to accomplish? Often I am able side step the issues of methanol with: gluderaldyhide fixation, 1% BSA in PBS incubation, then acetone permiblization. I understand that my "work around" does not work under all conditions, but it might be woth a try.



Hi all, ;)
Does anybody know if fixing and permeabilizing with methanol (with or without PF) generates artifacts? and the use of different serums to block?


Hi there,

I have used 4% formaldehyde prepared in PBS and it seems to work fine. I fix the cells with 4% formaldehyde for 8-10 min on ice and then continue further with the protocol.

Hope this helps

Pmaj

I've heard of both methods. I use the latter, since it's simple. But I heard if your working on cytoskeletom or membrane protein, the first method would be better. May it help you and good luck.
Je pense, donc je suis

#5 LostintheLab

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Posted 15 March 2009 - 08:51 PM

Hi,

Glad to see someone else interested in fluorescence work on this forum. Based on my work I have always found working with methanol to tricky. What kind of staining are you trying to accomplish? Often I am able side step the issues of methanol with: gluderaldyhide fixation, 1% BSA in PBS incubation, then acetone permiblization. I understand that my "work around" does not work under all conditions, but it might be woth a try.



Hi all, ;)
Does anybody know if fixing and permeabilizing with methanol (with or without PF) generates artifacts? and the use of different serums to block?


Hi there,

I have used 4% formaldehyde prepared in PBS and it seems to work fine. I fix the cells with 4% formaldehyde for 8-10 min on ice and then continue further with the protocol.

Hope this helps

Pmaj

I've heard of both methods. I use the latter, since it's simple. But I heard if your working on cytoskeletom or membrane protein, the first method would be better. May it help you and good luck.


Have you tried the different methods on the same cells/tissue (whatever you are trying to stain)?
If I fix with PFA (4%) I usually permeabilize my cells using 0.5% triton in a PBS/BSA/Na azide solution for 5 mins and then wash in PBS. As for blocking I usually do, using either goat serum or 0.5% fish gelatin in the PBS/BSA/Na azide, I find this gives clear staining. But its important to try different fixing methods for your cells because your staining will look different and it should help you identify potential artifacts. If you use PFA- fresh PFA is best and correctly made up as you will find the fixation is poor with old or poorly made up PFA and you may get more artifacts that way.

Intermediate filament staining works best with 1:1 methanol: acetone fixation for me anyway, PFA generally gives poor results with that, but is better for membrane associated proteins.
I knew it! I knew it! Well, not in the sense of having the slightest idea, but I knew there was something I didn't know.

#6 MMG

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Posted 17 March 2009 - 05:21 AM

Yes, organic solvent fixation sometime causes the loss of target, for example, the overexpressed GFP can not be retained by cold methanol fixation. And it may also cause the distortation of the target organelle. But for the staining of the proteins with high cytosol population or in the highly proteinaceous structures, organic solvent is better than the cross-linking chemicals, such as PFA and formaldehyde




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