RE: InSitu

From:"Plummer, Timothy B." <>


2 cents + 

Its very difficult to comment on this problem without knowing the
particulars of your specific protocol, but I will approach it generically.

Have you isolated the fact that this background is actually being caused by
the DAB reagent? ISH procedures are composed of several steps which can
contribute to background staining.  One can verify the DAB as the problem if
a slide is run through the entire protocol while receiving no probes or
detection chemistry.  Employing BSA or other blocking reagents prior to the
application of the pobe detection reagent is a necessary step in most cases.
Other potential areas to examine would be appropriate prehybridization
blocking, specimen acetylation, probe titer, detection reagent titration,
and of course, stringency variables.  Also, in the case where riboprobes are
employed (cRNA) one should consider the use of RNase A to remove
non-duplexed probe. 

 Having stated all of that, I will mention that I have also seen DAB give
muddy and nonspecific background patterns at timesin both IHC and ISH
procedures.  Switching to a different chromagen can improve this problem at
times, I recently used Nova Red from Vector laboratories and find it to be
an excellent reddish (not true red -maybe a brick red) chromagen for
peroxidase which is insoluble in organics.   Also inappropriately prepared
or differing formulations of DAB can create muddy backgrounds at times.


> -----Original Message-----
> From: [SMTP:Patsy.Ruegg@UCHSC.e
> Sent:	Wednesday, June 06, 2001 10:33 AM
> To:;
> Subject:	RE: InSitu
> blocking with acetylated bsa can help also using serum from the animal
> species you are staining (staining rat tissue, put rat serum in with the
> serum block which should be the host of the secondary), and i use the
> serum
> block before the primary and before the link.
> patsy
> -----Original Message-----
> From: Polkovitch, Debbie (PRI) []
> Sent: Wednesday, June 06, 2001 7:43 AM
> To: ''
> Subject: InSitu
> Does anyone know how to prevent background collagen staining when using
> with an InSitu procedure? 
> Thank you, 
> Debbie Polkovitch 
> RW Johnson, PRI 
> Spring House, PA 19477-0776 

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