[Histonet] eGFP Protein

[Gayle Callis]

Frances,

Yes, possible

What are you planning to do with the tissues being frozen-- either prefixing 
and cyroprotecting or cutting fresh frozen tissues?  How the tissue is 
handled initially will often determine seeing the eGFP.  For frozen sections 
here, including undecalcified bone frozens, if we fix a fresh FS with a 
solvent, we detect our GFP via immunofluorescent staining.  AntiGFP (either 
Goat or Rabbit - Rockland) and coming back with a secondary labelled with 
FITC or Alexa 488.  We have found our solvent fixation ruins the GFP, a well 
known problem.   Ig you are going  to use IFA staining for FFPE tissues, 
Teri Johnson prefers rabbit antiGFP instead of goat antiGFP - she says is 
has given her less background staining with the FFPE.  Since we do only 
frozen sections, we like the goat, antiGFP,  but either with work.  We buy 
our FITC conjugated secondaries from Jackson, usually made in Donkey, 
adsorbed to species being stained and also F(ab')2 frag of IgG whenever 
possible.

With formalin fixed tissues, it may better to detect the eGFP with antibody, 
and come back with a secondary labelled with either FITC or Alexa 488.  We 
always use a green fluorophore so it matches the color of eGFP.

We have cut unfixed frozen sections, let them air dry and NOT FIX but mount 
the dry section with Molecular Probes prolong gold with DAPI although we 
have used it without DAPI too.  These need to be looked asap, usually the 
same day for us.

We do NOT like PFA or NBF fixed tissues for frozen sections due to the 
aldehyde induced autofluorescence as this can mess up seeing a weak GFP 
signel.

There are ways to help reduce autofluorescence chemically.  Go to IHCworld 
website, and click on fluroescence and autofluorescence.  There is a link to 
a group in Toronto that is a superb discussion of autofluorescence and how 
to get rid of it.  For FFPE tissue, we have use the glycine method to remove 
free aldhydes.  It is simple and works well for our minimally fixed NBF 
tissue, no more than 8 hours, very thin pieces of tissue to guarantee good 
fixation.


Good Luck

Gayle M. Callis
HTL/HT/MT(ASCP)


----- Original Message ----- 
From: "Swain, Frances L" <SwainFrancesL <@t> uams.edu>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Wednesday, August 13, 2008 11:22 AM
Subject: [Histonet] eGFP Protein


One of my PI's is wanting to identifiy eGFP in not only frozen sections but
also paraffin sections. These will be mouse aortas is that possible?



Frances L. Swain HT(ASCP) A. A. S.

Special Procedures Technician

Department of Orthopaedic Surgery

Center for Orthopaedic Research

Barton Research Building 2R28

4301 West Markham Street

Little Rock AR 72205

(501) 686-8739 PHONE

(501) 686-8987 FAX

swainfrancesl <@t> uams.edu email




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