Tom,
Methanol kills GFP fluorescence. Just embed the cells in PBS-glycerol and
take your pictures.
Since your fluorescence already is inside the cell, you don't need methanol
for permeabilizing. Formaldehyde could reduce GFP fluorescence if incubated
too long, just for obtaining a picture it's unnecessary, too.
Wolfgang
At 10:43 PM 2002/11/4 GMT, Tom Nicholson wrote:
>Hello,
>>I was hoping someone could help me - I have made GFP constructs and
>transfected them into my cells. After sorting, the FACS means range
>from about 80 -100 (with a negative of about 3-4). However, when I take
>these cells to the fluorescence microscope, I do not see any
>fluorescence. I have been fixing my cells with 4% formaldehyde,
>followed by methanol treatment. I then add a 1:1 solution of
>PBS:glycerol, and seal with molten agarose. I visualize using the FITC
>filter on our microscope.
>>Any suggestions why I do not see anything would be greatly appreciated.
>>Thanks.
>>
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