Zeenobia,

I also get these punctate dots that are present on the negative controls with the TSA 
on cryosections.  I wonder if this is technique-specific.  Do people also get these 
punctate dots on paraffin sections?  The reason I ask is that the 
signal-to-background ratio with the TSA substrate is exquisite on whole mount preps, 
but absolutely lousy on cryosections.  My hypothesis is that the substrate has a high 
affinity for the lipophilic component of membranes, which it never sees in whole 
mounts, and would be washed away in paraffin sections.

Karen in Oregon



Date:          Tue, 28 Mar 2000 14:02:29 +1000
From:          Debbie Gae Pepperall <dpeppera@mail.newcastle.edu.au>
Subject:       Re: Signal Amplification
To:            Marsha R Price <mprice26@juno.com>
Cc:            histonet@pathology.swmed.edu

Hi Marsha

I noticed you mention the TSA...Have you used the CSA from Dako? I have
used it....but not impressed with it, as it picks up a lot of non-specific
staining...beautiful punctate dots on the membrane of cells in both the
positive and negative sections. I have contacted DAKO...they know of these
"unfortunate" staining patterns...however cannot give me an answer yet. I
have tried x-tra washing, blocking, avidin/Biotin Block you name it...even
diluting the antibody to infinitum. just thought I would throw this curve
ball into the mix. Hope we can overcome this.
Have any suggestions? Thanx in advance.
Cheers

Zenobia Haffajee
HAPS, Newcastle, NSW, "down Under".


At 08:25 AM 27/03/00 -0500, you wrote:
>Amos,
>have you heard of Tyramide Signal Enhancement? It works great and is easy
>to use.
>Marsha Price
>
>On Sun, 26 Mar 2000 09:14:54 -0500 amos brooks <atbrooks@snet.net>
>writes:
>>Hi,
>>    DAKO makes a Catalyzed Signal Amplification Kit. This amplifies 
>>the signal
>>enough to cause an antibody that usually doesnt stain on FFPE 
>>(formalin fixed
>>paraffin embedded) tissues. The procedure is a bit complicated and 
>>rather
>>expensive but the results can be quite quite good.
>>    Ventana also makes a one step amplification reagent that I've 
>>heard is good
>>although I've not used it much.
>>Amos Brooks
>>
>>Jay Turner wrote:
>>
>>> I am staining a fractured rat femur with an immunohistochemical 
>>stain to
>>> IGF-1.  The stain is a standard biotin-avidin peroxidase with DAB.   
>>The
>>> samples were decalcified in Cal-Ex (Fisher), it was a little harsh.  
>>There
>>> is stain in the section but it is very faint.  I don't want to re-do 
>>these
>>> animals and re-decalcify.  I have tried to use a metal enhanced DAB 
>>(cobalt)
>>>   but the color is too close to the hematoxylin counterstain.  Is 
>>there any
>>> other way to enhance DAB and the staining?  Any suggestions?  Is
>>> fluorescence the way to go?
>>>
>>> Thanks!
>>>
>>> ______________________________________________________
>>> Get Your Private, Free Email at http://www.hotmail.com
>>
>>
>>
>>
>
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