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Vibratome (~ 150um thick slice) to identify GFP signal in the tissues


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#1 wdyeo

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Posted 08 December 2012 - 12:04 PM

Dear All,


I injected GFP expressed cells into the mouse and i would like to identify where the GFP cells will be distributed/circulated in the normal mouse.


I would like to ask if I can use the vibratome (~ 150um thick slice) to identify GFP signal in the organs (lungs, heart, liver, spleen , pancreas, brain) which are fixed in 4% parafolmadehye?
Or i can PCR the organs to see if there's band for the GFP.
Which way is better to identify/detect the GFP cells or any other suggestions?


Thank you. Posted Image

wdyeo

#2 bob1

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Posted 08 December 2012 - 12:21 PM

You could use a vibratome, but you might be better off using a cryostat or regular sectioning using a microtome, especially as the tissues are already fixed in formaldehyde. Note that formaldehyde fixes often lower or remove native GFP fluorescence.

Either method will work well. However, the PCR will only detect the presence of DNA, unless you do some reverse transcription-PCR to detect transcript, the presence of DNA doesn't indicate that GFP would be expressed there. YOu could also do western blots on the tissues.

#3 wdyeo

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Posted 10 December 2012 - 03:21 AM

Dear bob1,
Thank you very much for the information and the suggestions.
You have been a great help Posted Image
Have a nice day.




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