Can I use 4% PFA fixed tissue for western blotting?

All Answers (7) Show full discussion

• 
  Jan Gebauer · Universität Köln

  My first thought was NO, as PFA is a fixative which crosslinks proteins. After a little bit of googling I am not so sure anymore, as this crosslinking is reversible. So you might give it a try. But why do you want to fix it prior dissecting anyway? For stability?

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  My first thought was NO, as PFA is a fixative which crosslinks proteins. After a little bit of googling I am not so sure anymore, as this crosslinking is reversible. So you might give it a try. But why do you want to fix it prior dissecting anyway? For stability?

  Aug 14, 2012
• 
  Arvind Shukla · Indian Institute of Toxicology Research

  Thanks for your suggestion. Actually the brain tissue is very delicate, and to find the specific portion of it there is need of fixation.

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  Thanks for your suggestion. Actually the brain tissue is very delicate, and to find the specific portion of it there is need of fixation.

  Aug 14, 2012
• 
  Deveroux Ferguson · Mount Sinai School of Medicine

  I don't think you should perform a western blot on the fixed tissue. The fixation process may produce artifacts which will complicate interpretation of your western. Alternatively, you can rapid freeze the tissue then perform a western blot at a later date. Secondly, you can simple perform an immuno for your protein of interest,

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  I don't think you should perform a western blot on the fixed tissue. The fixation process may produce artifacts which will complicate interpretation of your western. Alternatively, you can rapid freeze the tissue then perform a western blot at a later date. Secondly, you can simple perform an immuno for your protein of interest,

  Aug 14, 2012
• 
  Alicia Arenas ·

  I think you need incubate your sample at 90ºC for 20 minutes before the PAGE-SDS

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  I think you need incubate your sample at 90ºC for 20 minutes before the PAGE-SDS

  Aug 15, 2012
• 
  Ian Murray · Texas A&M University System Health Science Center

  I would not, as frozen tissue is better. You would likely not be able to publish the data, as the reviewers would most likely ask for frozen tissues. I am not sure as to the extent of the complete reversibility of the PFA crosslinks.

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  I would not, as frozen tissue is better. You would likely not be able to publish the data, as the reviewers would most likely ask for frozen tissues.
  
  I am not sure as to the extent of the complete reversibility of the PFA crosslinks.

  Aug 15, 2012
• 
  Ranjan Kumar · The University of Calgary

  I would recommend not to use PFA fixed tissue for western.

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  I would recommend not to use PFA fixed tissue for western.

  Aug 16, 2012
• 
  Ashish Misra · Yale University

  I know about mouse frozen sections, even if you fix with PFA 4% you can perform western blot. However, you need to do extensive washing (3X10 Min.) with PBS. I hope it will work for you.

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  I know about mouse frozen sections, even if you fix with PFA 4% you can perform western blot. However, you need to do extensive washing (3X10 Min.) with PBS. I hope it will work for you.

  Aug 20, 2012