[Histonet] Visualizing dual endogenous Fluorescent Proteins in mice
tissue (fresh or fixed)
zhengl02 <@t> student.ucr.edu
Fri Aug 4 03:42:46 CDT 2006
Hey, I am new.
Got a newbie question.
I have shed much blood searching for info on how to visualiz these:
transgenic mice tissue with endogenous mRFP1 (monomeric RFP by R.Y Tsien) in cytoplasm AND eGPF on cell membranes.
Currently, I fix the fresh tissue(thin sections) in 4%PFA followed by cryosections. The mRFP1 look good, but eGFP channel is filled with autofluroescence (by PFA I guess).
Using NaBH4 did not help with eGFP.
I am really dying for a protocl that could:
1) Maximize the eGFP intensity against background
2) Minimize the green channel autofluorescence
3) Maintain the membrane localization of eGFP so that cell morphology can be kept
4) No immunostaining can be used (rule of my game...), must be FP-derive fluroescence
I read much and notice several taboos:
1) no organic solvent in the processing (kills FPs)
2) NaBH4 may help (actually not for me)
3) Zinc buffer (no formalin) may help maintain membrane bound FP? antigen?
4) Fresh tissue snap frozen then sectioned yields good eGFP?
I am very lost and bombarded with information from lots of people. If you are a "mice people" working with endogenous eGFP, can you throw me a straw please?
The key things here are the mRFP has no problem, but the eGFP is membran-bound and maybe of low level.....and I need to be able to see that eGFP!!!
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