[Histonet] Immunofluorescence staining/minimizing background staining

Kim Merriam kmerriam2003 <@t> yahoo.com
Wed Feb 1 08:45:24 CST 2012

What fluorochromes are you using?  There is a lot of autofluorescence in the FITC channel.  Have you looked at an unstained tissues under the scope with each filter that you need, that may give you a clue as to where your background is coming from.  In addition to an unstained slide, I suggest eliminating the primary to see if your secondary is sticking to the tissues.
Are you doing single-color or double stains?  If you are getting a lot of autofluorescence with one fluorochrome, I would suggest trying a different one.  We use alexafluur-647 (far red) a lot because is it fairly clean (ie - very little autofluorescence in that channel)

Kim Merriam, MA, HT(ASCP)QIHC
Cambridge, MA

From: "Kasai, Miki (NIH/NCI) [E]" <kasaim <@t> mail.nih.gov>
To: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu> 
Sent: Monday, January 30, 2012 4:50 PM
Subject: [Histonet] Immunofluorescence staining/minimizing background staining


We are performing some immunofluorescence staining on mouse lung tissue.  We
are getting some nice positive staining with some of our initial antibodies
(procollagen, cytokeratin).

We would like to minimize the amount of background staining we are getting.
We are titering our primary antibodies to find out optimal Ab concentration
as well as the secondary conjugate Ab with the fluorophore of interest.  We
use donkey serum for general blocking.

Any other suggestions?

Much appreciation,

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