[Histonet] peptide getting into tumor cells

From:"Elizabeth Chlipala"

Hello everyone
 
I have a unique question.  I'm working with a client who is trying to
duplicate some previously published work.  They have a peptide that is
supposed to target tumor cells (internalized by tumor cells). What they
are looking at is targeted drug delivery.  In publication they took
unfixed frozen sections of human tumors and incubated them with a FITC
labeled peptide for 12 hours at room temperature in a humidity chamber
and then rinsed in PBS with glycine for 48 hours with multiple changes.
The materials and methods in the publication were very minimal.  The
client followed their staining protocol but used both fixed and non
fixed frozen sections.  The staining pattern was diffuse.  I made some
initial suggestions of looking at fixed unstained slides and then a FITC
labeled peptide that had been scrambled (not the same sequence) using
the same method.  My question is about the technique that is being used.
I have done a lot of immunohistochemistry and immunofluoresence but the
concept here is a bit different in that we are trying to determine if
this peptide actually is internalized by tumor cells.  To me the
incubation time seems long (these are 4 micron sections) and they did
not use a blocking reagent to avoid charge related binding.  If anyone
has done anything like this before and has any suggestions that would be
great.  
 
Thanks in advance.
 
Liz  
 
Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
P.O. Box 18592
Boulder, Colorado 80308
Office: (303) 735-5001
Fax: (303) 735-3540
liz@premierlab.com
www.premierlab.com
 
Ship to Address:
Premier Laboratory
University of Colorado
MCDB, Room A3B40
Boulder, Colorado 80309
 
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