RE: [Histonet] IHC losing tissue

From:"Jeff Gordon"

Cell Marque promotes using pressure cookers with our Trilogy EDTA solution, which is a pretty aggressive pretreatment.  We use this with pretty much all of our tissues, including breast and prostate needle biopsies and skin and other sensitive tissues.  To do this, it is essential to prepare the cut tissue properly on the slides to ensure that no tissue is lost.  In some cases, if you are using higher pH solutions for heat retrieval, the tissue preparation still can't prevent the tissue from coming off, which is why oftentimes people will move from high pH solutions to Trilogy, which doesn't have the higher pH but still gives comparable staining.
This is our standard method of slide preparation:
1) Cut tissue 3-4 microns.  This is sufficient for clinical IHC.  Anything thicker runs a much higher risk of tissue loss.
2) Use positively charged slides that have been handled carefully (only touch the label area or the edges, do not touch the face of the slide as it may draw the charge off the slide).
3) Dry the slides for at least 2 hours at 58 degrees Celsius in a slide drying oven.  For fatty breast, extend the drying time to 4 hours.  This ensures that all water will escape from underneath the melted paraffin and tissue, and allow the tissue to form an uninhibited bond with the slide.  If you have to dry the tissue faster, you can also use a microwave method.  Methods used with the microwave for slide drying include running the microwave for 1.5 minutes, then allowing it to sit for one minute for the paraffin to congeal, and then run it for another 1.5 minutes, and then let sit again for a minute.  The slide is ready for heat retrieval now and should give you good tissue adhesion.
By using that method, we don't have to use any additives in our waterbath for tissue adhesion.
Jeff Gordon
Cell Marque Corp.

	-----Original Message----- 
	From: on behalf of 
	Sent: Wed 1/18/2006 1:47 PM 
	Subject: [Histonet] IHC losing tissue

	About your IHC slides losing tissues during processing: It's probably what
	Rene and Linda said - preheating is necessary. But I have heard that the
	treated slides purchased from vendors lose their stickiness over time. You
	might check other lots or other vendors to see if you still have problems.
	Were these slides brand new? Has anybody else had problems with old
	pretreated slides losing their stickiness?
	Melville B. Vaughan, Ph. D.
	Assistant Professor
	Department of Biology
	University of Central Oklahoma
	Edmond, OK 73034
	Histonet mailing list

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