Posted by
Phillips, Thomas E. on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Blocking-in-immunocytochemsitry-ImageIT-FX-Enhancer-tp7580055p7580063.html
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Jason - Did you accidently invert the charges in your reply - according to Invitrogen's website "Alexa Fluor(r) dyes and many other dyes have positively charged modifications..." and usually when one sees high background binding to the nucleus, it is assumed the negatively charged nucleic acids are the culprit. Tom
Thomas E. Phillips, Ph.D
Professor of Biological Sciences
Director, Molecular Cytology Core
2 Tucker Hall
University of Missouri
Columbia, MO 65211-7400
573-882-4712 (office)
573-882-0123 (fax)
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http://biology.missouri.edu/people/?person=88http://www.biotech.missouri.edu/mcc/-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Kilgore, Jason
Sent: Thursday, March 21, 2013 11:18 AM
To:
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Subject: Re: Blocking in immunocytochemsitry - ImageIT FX Enhancer? **vendor reply**
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** Vendor Reply **
Hi, Christophe,
I actually helped develop that product. The Image-iT FX Signal Enhancer solution isn't meant to replace protein blocking, and won't work as well as a substitute. Most dyes have a negative charge, which makes them nonspecifically attracted to positively-charged areas on cells and tissues (in cultured cells, that's mainly the nuclei and mitochondria. Brain tissue cryosections, it's the myelin). The Signal Enhancer blocks the positive charges. But you should still do a subsequent protein blocking. Not everyone needs it, but if you see background with a secondary-only control that includes a protein blocker, then you might want to add it as a step. I got to where I used it with every ICC / IHC protocol.
I've never found gelatin to be very effective in my assays as a protein blocker. I generally recommend 6% Bovine Serum Albumin / 10% normal serum / PBS. Another commercial option (one of those "proprietary" things you avoid) is called BlockAid, from Life Technologies, which is a mix of various protein blockers. Use it undiluted or lightly diluted as a blocker and in with your primary. I've found it to be as good or better than any other common blocking option I've tried for ICC / IHC (even though we market it for blocking microspheres).
Cheers,
Jason
Jason A. Kilgore
Technical Application Scientist
Molecular Probes Labeling and Detection Technologies Cells Systems Division
T 1 800 955 6288 then option 4, then option 6, or 541 335 0353 * F 541 335 0238
29851 Willow Creek Rd * Eugene * OR * 97402-9132 * United States www.invitrogen.com/technicalsupport
-----Original Message-----
From: Confocal Microscopy List [mailto:
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Sent: Thursday, March 21, 2013 2:24 AM
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Subject: Blocking in immunocytochemsitry - ImageIT FX Enhancer?
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Hi,
Not a confocal question, but a lot of people are doing immuno here so I thought I'd ask. We are currently using gelatin as the main blocker in our imunocytochemistry protocol. I was wondering if we could get better staining (less background / less non specific labeling) by switching to something else and found Life Technologie's ImageIT FX Enhancer (
http://products.invitrogen.com/ivgn/product/I36933). Does someone have experience with this, and is the added cost justified? I try to stay clear of proprietary formulas but sometimes (see Prolong Gold) a "secret" product does work better.
Thanks for the advices,
Christophe
--
Christophe Leterrier
Researcher
Axonal Domains Architecture Team
CRN2M CNRS UMR 7286
Aix Marseille University, France