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GFP immunohystochemestry PFA issue


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6 replies to this topic

#1 tiziana

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Posted 30 September 2010 - 11:11 PM

Ciao,

I need to performe an immunohistochemistry for eGFP detection in not perfrused brain (just frozen in dry ice or fixed soon after been cut and stored a 4 C) and I wish not to eluite my protein after the first wash :(

any suggestion???

Cheers
tizzy :D

#2 bob1

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Posted 02 October 2010 - 02:06 PM

The GFP should glow quite nicely under a fluorescent microscope. You shouldn't need to do any IHC on these slides.

#3 tiziana

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Posted 03 October 2010 - 09:50 AM

....actually I do...I injected lenti_eGFP at the beginning of the RMS and 2 weeks after I cut and looked at the microscope to see migrated neuroblasts into the Olfactory Bulbs...and they were there, I mean the signal was there but it was not possible to do any cell quantification.
It was like to see a green signal very specific but with very bad green cell morfology :-(

At that point the sections have been frozen but when I de-frozen and fixed them with PFA I found my green much weacker :-( at the moment I'm planing to do an immuno to signail retrive but I do not have much hope to have the resolution I need for the quantification...pre fix brain with PFA or much warse perfuse, make the tissue very difficult to be cut with the criostat, and I scared to loose my samples.....I guess there is no much to do....

#4 bob1

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Posted 03 October 2010 - 03:18 PM

I think you are having some fluorescence issues. After fixing try washing a few times for 1 hour each in 0.1 M glycine in PBS/TBS at room temp or in the fridge. It should cut down the fluorescence from the fixing.

For autofluorescence try fixing in DMSO/methanol and then clearing in DMSO/Methanol with 2% H2O2.

#5 tiziana

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Posted 04 October 2010 - 05:41 AM

Many thanks .....I will try to cut down the fluorescence an you suggested
and see if the cellular resolution get better :rolleyes:

about the fixation in DMSO/methanol:
1. I supposte DMSO is to crioprotect and methanol as fizative, you meant I should fix my brain in this way, freeze it and then cut it ???

2. is this tretment suitable for GFP detection? I mean because of
the small size of the protein I thought the best choice would be PFA fixation
(because of the reticulum it forms over the proteins to block
them were they are) but I really don't know why methanol would has fixation property.....by the way do you think this would be strong enought for GFP???

Cheers
tiziana :ph34r:

#6 bob1

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Posted 04 October 2010 - 03:21 PM

Dent's fixative is the DMSO/methanol (80%/20%) mix commonly used for this sort of thing. It should be fine for GFP I would have thought. I'm not sure about cutting with it, I;ve only used it on whole mount embryos.

#7 tiziana

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Posted 05 October 2010 - 12:05 AM

ok...thanks...I will try both :rolleyes :




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