Dear Dr. Scott,

Thank you very much for your kind suggestions!

Sincerely,
Yichao WU


>From: "Turner, Scott" 
>To: "'yichao wu'" 
>Subject: RE: How to do double fluorescent staining with two Monoclonal pri  
>   mary antibodies?
>Date: Tue, 1 Jul 2003 14:21:17 -0400
>
>One thing you might try is preincubating a Fab fragment secondary antibody
>conjugated to FITC with primary A and preincubating primary B with a Fab
>fragment secondary antibody conjugated to Texas Red.  The method is as
>follows: incubate primary and secondary to form a complex in a test tube
>prior to application on tissue.  Then add serum from host animal (in this
>case mouse) to the tube to block unbound Fab.  Then apply the complexes to
>the tissue.  Working out the right concentrations of secondary and serum is
>the biggest hurdle to this approach.  Fortunately, this is essentially how
>the DAKO Animal Research Kit  (ARK) works, and I believe that they make a
>FITC version.  For the Texas Red, you could use the biotin ARK then come
>back with a Texas Red labelled streptavidin tertiary reagent.  In theory,
>this should solve your problem, though working out all the details might be
>a bit tricky.  The biggest problem with this is that the kits are fairly
>expensive, and you'd need to buy both kinds of kits (FITC and biotin) to
>make it work.
>
>Scott Turner
>DNAX Research, Inc.
>Palo Alto, CA
>
>-----Original Message-----
>From: yichao wu [mailto:yichaowu@hotmail.com]
>Sent: Monday, June 30, 2003 10:11 PM
>To: histonet@pathology.swmed.edu
>Subject: How to do double fluorescent staining with two Monoclonal primary
>antibodies?
>
>
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>Dear ALL,
>
>Unfortunately I must do double fluorescent immunostaining with two mouse
>monoclonal primary antibodies now. That is to say ,the primary antibodies
>are from the same animal origin as follows,
>
>Mouse anti-A monoclonal antibody and Mouse anti-B monoclonal antibody.And
>the secondary antibodies are conjugated with FITC and TRITC respectively.As
>I know, these secondary antibodies could not differentiate IgG subclass.
>
>My objective is to show the primary antibodies on one section 
>simultaneously
>with secondary antibodies as above.
>
>SO I meet with the most difficult type of double immunostaining. To avoid
>cross-reaction between the secondary antibodies and the primary antibodies,
>I should try my best and long for your help as well.
>
>I've learned and tried the method of "post-fixation with paraformaldehyde
>steam". After incubation with the first kind of secondary antibody, 
>incubate
>the section in PFA steam for several hours to inactivate the first kind of
>secondary antibody. Then add the second kind of primary antibody.
>
>I succeeded once. But to be frankly, it is hard to control. I failed many
>times later. In my neighbor lab, a supervisor could manage this kind of
>double immunostaining well. Between incubation of the first kind of
>secondary antibody and the second kind of primary antibody, she use a kind
>of #161##176#Acidic Solution for antigen retrieval#161##177# to incubate
>section with application of microwave oven for 10 minutes.(But it is not
>citrate buffer as I know. And I#161#Ove tried citrate buffer and
>failed).However she would never tell me what components it has.
>(SAD.......it is one of her secrets).
>
>I tried to find such a kind of acidic retrieval solution which could
>inactivate secondary antibody and retrieve antigen as well.Is there any 
>kind
>of retrieval solution whose PH may be very low.And could be reused for many
>times as I know?
>
>I wonder if you have such experience of double immunostaining? If so, could
>you kindly give me some advice?
>
>Thank you very much!
>
>
>
>Yichao WU, Ph.D Candidate
>
>Email: yichaowu@hotmail.com 
>
>Research Institute of Nephrology
>
>Jinling Hospital
>
>Nanjing 210002 China
>
>
>
>
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