Methods and Techniques Discussion => Electron Microscopy => Topic started by: leahf on January 16, 2005, 08:27:51 AM

Title: Is PFA-fixed tissue usable for TEM?
Post by: leahf on January 16, 2005, 08:27:51 AM
Hi All
 I've tissue samples originally fixed for FISH, which i would very much like to use for transmmision electron microscopy. my problem is, does my fixation fit TEM?

i had fixed for 20 hours in 4% formaldehyde at 4 degrees, then washed away  the formaldehyde with PBS and kept the samples at 1:1 ethanol:PBS , at -20 degrees, for the past 4 months.

I've been told, for TEM, that gluteraldehyde fixation is best and that samples should be kept at 4 degrees in glutaraldehyde buffer.
 
will my samples be usable anyway? if not, is there a way to "fix" this problem? (short of getting fresh samples, which i wont be able to do till summer...)
Title: Is PFA-fixed tissue usable for TEM?
Post by: excalibur on January 16, 2005, 12:55:47 PM
Your samples are salvageable, although not optimal. The next step for TEM is postfixation in osmium tetraoxide. Then processing steps to the plastic infiltration for embedding & sectioning.
Title: Is PFA-fixed tissue usable for TEM?
Post by: immunoem on January 17, 2005, 04:49:23 AM
As Paula says, you can process the samples on for TEM. However, PFA alone is not recommended for EM studies due to poor ultrastructural preservation. I would also be worried about the storage at -20oC, especially if the samples were not cryoprotected.  The best thing would be to resin-embed a couple of samples and see what they look like. You never know until you try.
Good luck.
Ian
Title: Is PFA-fixed tissue usable for TEM?
Post by: leahf on January 18, 2005, 06:06:46 AM
OK, will try. Thanks for the encouragement!