[Histonet] RE: Histonet Digest, Vol 128, Issue 17

[Riesen, Rebecca]

Liz had some great suggestions! The one that I have found to be the MOST important is #4.  Standing my slides up at a slight angle and allowing the moisture to drain out from under the section AND then letting the drained slides sit and dry for a good long time is extremely important for adherance to be successful (I've never gone as far as a week, although it couldn't hurt, but at least overnight seems necessary).  I would focus on that, so you first get good adherance and then the other tips following the proper drying will then help keep them on.  Good luck!

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Message: 4
Date: Fri, 18 Jul 2014 14:44:13 +0000
From: "Murphy, Valerie" <murphyv <@t> karmanos.org>
Subject: [Histonet] Brain tissue coming off slide
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We are staining human FFPE brain sections for Her 2 Neu and we're having a problem with tissue coming off the slide during the HIER step.
We are currently using Vectabond coated plus slides and using a decloaking chamber for HIER.
Are there any other steps we can take to prevent  tissue coming off the slide?

Thank you,

Valerie Ratliff
Karmanos Cancer Institute


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Message: 5
Date: Fri, 18 Jul 2014 09:16:04 -0600
From: Elizabeth Chlipala <liz <@t> premierlab.com>
Subject: [Histonet] RE: Brain tissue coming off slide
To: "Murphy, Valerie" <murphyv <@t> karmanos.org>,
	"'histonet <@t> lists.utsouthwestern.edu'"
	<histonet <@t> lists.utsouthwestern.edu>
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Valerie

There are a few things that you can try.

1.  This may or may not work with the Her2 IHC, since for that antibody I believe it requires retrieval at least at 98C but time and temperature are related with respects to heat retrieval.  You could try 70C for 2 hours this is gentler on the tissue and works for some antibodies but not all of them.  For example it does not work for Ki-67 in our hands.  We use this retrieval method on bone and other types of friable tissue if necessary.  

2.  We have found that some samples after HIER may require manual staining methods in order to optimize tissue adherence, not sure if that is an option for you too.

3.  Post fixation in 10% NBF after deparaffinization for about 10% minutes.  This may help tissue adherence but we have also found that this may decrease the sensitivity of the protocol and you may have to adjust your primary antibody concentration.

4.  Tissue adherence to the slide especially with brain (we have seen this on rodent brains) requires proper fixation and adequate processing, poorly fixed or processed brain samples will have issues with tissue adherence.

5.  Allow the cut slides to sit for about a week prior to staining we find this may help with tissue adherence.

Good Luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
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liz <@t> premierlab.com
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