Re: immuno staining

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From:"Jay Turner" <turnerjayd@hotmail.com>
To:dpeppera@mail.newcastle.edu.au
Reply-To:
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Thanks for your reply Zenobia.

We're actually not using HIER or enzyme digestion at all for our antigen 
retrieval with the growth factor immunos.  We've been using a retrieval 
solution from BioGenex that is made specifically for acid-decalcified bone 
samples, which we have had good luck with in the past, and are blocking with 
3% powdered milk in 1% BSA.

I don't think the nuclear staining that I mentioned, which shows up in 
addition to the expected cytoplasmic stain, is a result of over-retrieval.  
Similar results have been reported in articles I have found and appear to be 
accepted, I just can't seem to find an explanation as to why this might 
occur.  Could IGF-1 have nuclear receptors, which could explain this nulcear 
staining?  Any ideas?

Thanks in advance.

Jay Turner
Department of Orthopaedics
University of Medicine and Dentistry of New Jersey
Newark, NJ 07103
Phone: 973-972-4408
Fax: 973-972-5294

>From: Debbie Pepperal <dpeppera@mail.newcastle.edu.au>
>To: Jay Turner <turnerjayd@hotmail.com>
>Subject: Re: immuno staining
>Date: Fri, 07 Apr 2000 14:24:55 +1000
>
>Hi Jay
>  Just as a thought, are you pretreating your sections using microwave
>retrieval ? Sometimes the tissue tends to get overdigested, opening up more
>antigenic sites than necessary. Have you tried blocking steps; normal serum
>or even an avidin / Biotin block? My 2 cents worth; hope your results are
>better.
>
>
>Zenobia Haffajee
>HAPS, Newcastle, Australia
>
>At 02:04 PM 03/04/00 -0400, you wrote:
> >I am running an immuno. stain to insulin-growth factor 1 in the fracture 
>of
> >a rat femur.  I finally have good results and the stain looks nice.  I am
> >using DAB and I am getting some of the cells stained in the nucleus.  I 
>have
> >seen this before in some old references.  IGF-1 should be staining in the
> >cytoplasm or matrix, is there a reason why IGF-1 would stain in the 
>nucleus?
> >  The pathway doesn't appear to create this situation, does anyone know 
>why
> >this might occur?
> >
> >
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> >

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