problem with fresh frozen eGFP sample

Posted by Jean Brennan on
URL: http://confocal-microscopy-list.275.s1.nabble.com/problem-with-fresh-frozen-eGFP-sample-tp593098.html

Hi all,

 

I have an animal that is Tg eGFP for a neurotransmitter in astrocytes.  It shows up very well in brain and spinal cord after perfusing with 4%PFA.  I'm trying to Laser capture some cells for RNA work but having trouble visualizing the cells in the fresh frozen tissue.  I'm doing cervical dislocation, the disection is under 1min., then placing the tissue into 2-Methylbuane that has been cooled and surrounded by dry ice.  The tissue is then cut in a cryostat, 8um section placed onto a slide.  I then visualize using either laser or mercury bulb (with 488 filter) and the cells look abstract or green everywhere instead of dark background with green astrocytes (star shaped cells).  I have cut frozen sections from PFA perfused animals several times and there is no problem with the way the cells look.  Do you think I need to hydrate the slide to visualize the cells better?  Is the freezing method faulty?  Any suggestions?

 

Thanks for your help.

Jean

 

 

Jean Brennan
Research Specialist
Rothstein lab, Dept of Neurology
Johns Hopkins University
600 N. Wolfe St., Meyer 6-174
Baltimore, MD  21287
410-614-4119; FAX: [phone]
[hidden email]