Methods and Techniques Discussion => Electron Microscopy => Topic started by: barbara999 on June 01, 2004, 09:14:29 AM

Title: EM for myelin
Post by: barbara999 on June 01, 2004, 09:14:29 AM
I am processing EM for myelin, however, the ultrastrure of myelin seemed to have some problems, that is the myelin of large axons seemed uncompact.  Does anyone know what should be responsiblr for the problem?? I need good suggest very much:(
Title: EM for myelin
Post by: richard03 on June 03, 2004, 03:54:01 PM
The tissue may not been fixed well.

Richard
Title: myelin
Post by: barbara999 on June 08, 2004, 07:44:24 AM
Thanks for your reply, Richard. I used 2%PF +3% GA as perfusion buffer. And I enenly try higher dilution of GA, but it still does not work.
Title: EM for myelin
Post by: richard03 on June 08, 2004, 10:29:14 AM
I normally use the opposite, that is higher conc of PF (4%) and lower conc of GA (1%). The reason is due to PF's penatration rate is faster than GA's.

Also you may want to check your dehydration steps to make sure the tissue is fully dehydrated.

Good luck!

Richard
Title: EM for myelin
Post by: barbara999 on June 09, 2004, 06:14:21 AM
Thanks a lot for your reply. I put the sections(less than 1mm) into the serials of ethanol for 10 mins each, and then the aceton two times, each 10 mins. Do you think it is not enough to fully dehydrate? And may i use the 4%PF + 3%GA?
Title: EM for myelin
Post by: richard03 on June 09, 2004, 01:22:25 PM
The differences from my protocol are
1) 15 min for 100% ethanol, two times;
2) 15 min for propylene oxide (instead of acetone), two times.
3) Fixative: 4%PF+1%GA in 0.1M PB, pH7.2

If problem persists, that may be what is suppose to be in tissue itself.

Richard
Title: EM for myelin
Post by: barbara999 on June 18, 2004, 12:34:48 AM
Thanks a lot!  Richard.