[Histonet] RE: Problem of Double immunoflouresence staining ..

[Melissa Gonzalez]

Sohail, 
First, Perhaps you should try incubating each primary with respective
secondary only, to determine how they look separately, and also make
sure each is working on its own. For example, you may not have your aSMA
dilution correct. Also, do you have a positive control for TRITC to make
sure you can detect any TRITC signal adequately? Once you have
appropriate signal from both, you can then make your cocktails. Also, in
the future, I would recommend switching to Alexa Fluors from Molecular
Probes, they really are great. In your instance you would use AF488
(FITC) and AF 546 or 555 (TRITC). But that is just my 2 cents. 

Good luck,
Melissa



Message: 4
Date: Fri, 29 Sep 2006 13:06:27 -0700 (PDT)
From: sohail ejaz <sohail_e <@t> yahoo.com>
Subject: [Histonet] Problem of Double immunoflouresence staining of
	retinal	vasculature
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <20060929200627.949.qmail <@t> web39506.mail.mud.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1

Hello everybody
   
  I have a big problem of staining retina vasculature using a cocktail
of two different antibodies. The problem is that i can only see staining
with FITC but not with TRITC, hence i cant make a merger of both FITC
and TRITC. 
   
  Cocktail of primary antibodies include (1) Monoclonal Rabbit Anti
human vWF (2) Monoclonal mouse Anti alpha SMA and cocktail of secondary
antibodies include (1)  Anti rabbit FITC (2) Anti mouse TRITC
   
  Please let me know your commentc to slove this issue.
   
  Dr.Sohail