[Histonet] HIER

[Merced Leiker]

In our experience, the heat is definitely more important...we retrieve 
epitopes ranging from cytoskeletal markers (Troponins, Myosin Heavy 
Chain,...) to nucler (PCNA) and others (c-kit, beta-catenin, vonWillebrand 
Factor-vWF). We use a steamer set to 30', just place the slides in flat on 
the bottom of the basket: encircle the sections with Pap pen first, then 
pipette some buffer on. I always test pH 6 and pH 9 but haven't seen a 
difference yet between the two with the epitopes we're unmasking.)

We have this wicked vWF antibody from Calbiochem (PC313) that unmasks in 
PBS (pH7) in the steamer and binds very well on both paraffins and frozens. 
(And it does need retrieval - without retrieval it is gives very little 
stain).

Ok, I digress!! But you get the point...that's from our experience, anyway!

~Merced

--On Monday, March 02, 2009 7:52 PM +0100 Casper Hempel 
<casperhempel <@t> gmail.com> wrote:

> Hi histonetters
> We have been talking a lot about how to retrieve your epitopes using a
> microwave in the best possible way.
> We haven't come to an agreement and people in my lab both argue that
> temperature and pH are important issues. Without a doubt both factors are
> important for a proper retrieval, but if you have to focus on one of the
> factors, which would you consider the most important? Temperature or pH?
> The issue is mainly longer incubations of the slides in boiling buffer.
> The buffer is evaporating and the solution/buffer gets less and less pH
> neutral and you need to top up with dH2O. However, if you boil with
> reduced intensity, less evaporation will occur and the pH will remain
> more stable. Do you have any suggestions or comments to this issue?
> Looking forward to your replies.
> Cheers
> Casper
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>



Merced M Leiker
Research Technician II
354 Biomedical Research Building
School of Medicine and Biomedical Sciences
State University of New York at Buffalo
3435 Main St, Buffalo, NY 14214
Ph: (716) 829-6033
Fx: (716) 829-2725

No trees were harmed in the sending of this email.
However, many electrons were severely inconvienienced!