Dear all, Thank you for the helpful ideas and suggestions. I've been asked to summarize what I've learned. 1. Why is the GFP fluorescence lost after exposure to fixation/ detergent? Different opinions here: *Formaldehyde destroys GFP. I measured GFP+ cells after fixation with 4% paraformaldehyde (BD Cytofix) compared to fresh GFP+ cells and there was no difference in my hands. I've seen past postings to this forum suggesting that formaldehyde can reduce GFP fluorescence so likely it depends on the quality of the paraformaldehyde and how freshly it is made (see this posting http://www.cyto.purdue.edu/hmarchiv/2006/1440.htm). * The detergent likely alters GFP's structure enough to reduce fluorescence. * GFP leaks out of the permeabilized cell. There are some previous reports clearly showing that membrane targeted GFP is retained following fix/perm procedures so it would stongly point out at a leakege rather then fluorescence attenuation (see Cytometry 29:286-291 (1997)). With regards to cytokines remember that you are crosslinking them not only in cytoplasm but also in the ER-Golgi network (therefore you often use Brefeldin A; BFA to inhibit vesicular transport and increase signal). As a result you have much less cytoplasmic GFP then cytokines in cytoplasmic + intercellular compartments. Two main suggestions here, either sort fresh cells for GFP+ and GFP- and then do the intracellular stain OR use an anti GFP antibody to determine if the GFP is leaking or just reduced in fluorescence. 2. Alternative reagents to preserve the GFP fluorescence One person suggested using Caltag Fix&Perm as they have seen no loss of EGFP fluorescence after permeabilization. FYI- Caltag is now owned by Invitrogen. Thanks again for your help. Kendra _________________ Kendra Hyland, Ph.D. Discovery Genomics, Inc. 614 McKinley Place N.E. Minneapolis, MN 55413 ph/ 612-656-4485 (office) ph/612-379-2956 x 6566 (lab) fax/612-379-6580 kendrah@discoverygenomics.net http://www.discoverygenomics.net/Received on Wed Dec 12 12:38:00 2007
This archive was generated by hypermail 2.1.8 : Wed Jan 31 2007 - 03:12:00 EST
