[Histonet] IHC ~ Tissue Falling Off Slides, drying procedure.slides

Fri Dec 10 09:54:55 CST 2004

Laura,

What type of tissue are you using?  Are you using superfrost plus or other charged slides?  I have been told that these have a shelf-life and after a certain amount of time (~ 18 months or so), the charge goes away.  This might be something to look into.

Kim Merriam
Novartis
Cambridge, MA

Jeff Gordon <JGordon <@t> cellmarque.com> wrote:
Laura, here are the issues that we deal with most in regards to tissue adhesion:

1) Drying (most common). When you room temperature dry the slides, the water will dry from the outside in. Once the water is out from the edge of the slide, the paraffin sort of adheres itself to the slide around the tissue, creating a seal that doesn't allow any more water to escape from under the paraffin. It can sit there for a month and still have water trapped under the tissue in this condition. When you put the slides in the oven, the paraffin melts allowing water to escape from under the seal and under the tissue, thereby allowing the tissue to fully adhere to the slide without any water barrier separating any part of it. Since you are already drying for 2+ hours, I would recommend just putting them in the 60 degree oven for that entire time. If you dry in the microwave, we usually dry for a total of 3 minutes under power. We run it for a minute and a half at full power, then let it set for 30 seconds to a minute (enough to allow the paraffin to solidify again) and then run it
 for another minute and a half and let it cool again.

2) Retrieval pH. The higher pH solutions are harsher on the tissue than other solutions, mainly because raising the pH will literally eat the tissue up (think about what Drano would do to your skin, but on a much lesser scale). That is where we usually promote Trilogy. It usually isn't as harsh as high pH (9 or 10) or straight EDTA because of the emulsifiers in it that help bring the pH to a more tolerable level for the tissue. Many labs that have used Trilogy brought it on board because of the harshness of other solutions that they were buying or making themselves. The more basic that you make the solution, the more likely it is that you will have tissue/morphology problems, regardless of the slide preparation.

3) Waterbath contaminates. Anything in the water can affect the adhesion of the tissue. Deionized water is preferred.

4) Tissue thickness. We never recommend that tissues cut at more than 4 microns be used for clinical IHC. We cut ours at 3-4 microns standard regardless of the tissue. If you cut them thick they are more likely to lose adhesion and can give background, as well.

5) Slide handling. Don't touch the flat surface of the slides or you can draw the charge off of them. Hold them by the label area.

6) Formalin pH. This is often taken for granted, but can have a major affect on tissue adhesion. If the formalin has too many hydrogen ions (is too acidic), it will pass a positive charge onto the tissue that it is fixing. Not good. The negative charge of the skin is what makes it adhere to the positive charge of the histology slide. If the tissue carries positive charge due to acidic formalin, and the slide is positively charged, then you know what like charges do. Try to keep the formalin as close to neutral pH as possible.

7) It is possible that a certain lot of slides that you receive could be bad. It happens to everyone sometime.

8) Retrieval time. We recommend using a pressure cooker, and one major reason is the speed. There are different suggested protocols for heat retrieving in a pressure cooker, and different pressure cookers on the market. Regardless of the suggested protocol or the brand of pressure cooker that you use, pressure cookers have one thing in common: they are faster than steamer methods. Pressure cookers reach a higher heat, and they are more aggressive than other pretreatments, but you have the slides in the hot solution for a much shorter time, which can be key. YOu even recognize that from reducing your steamer retrieval time from 75 minutes to 20 minutes in order to try and improve the adhesion. By using a pressure cooker (whether it is from us or from a competitor or from a kitchen store) you can improve your stains, increase your turnaround time, and also possibly help your tissue adhesion due to the short period that the slides are in hot solution (with Trilogy we don't require a
 cooldown period after heat retrieval). This is NOT a pitch for our pressure cooker. We don't care what pressure cooker you use, but we do recommend using the pressure cooker for your heat retrieval.

I don't expect any of this information be news to anyone on here. You all have been in this industry at least twice as long as I have been, but I try to help troubleshoot often and these are the factors that play into tissue adhesion questions that I try to help workout for clients. These are just reminders of things to check if you have an adhesion problem. I hope that this helps.

Jeff Gordon
Cell Marque Corp.
1-800-665-7284, Ext. 12

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of Jones,
Laura
Sent: Thursday, December 09, 2004 10:44 AM
To: Histonet (E-mail)
Subject: [Histonet] IHC ~ Tissue Falling Off Slides, drying
procedure.slides

Hey Jeff! We have tried many variations on drying as well. Generally, we
cut the tissue, let it stand and drain/air dry at room temp for 0.5-1.0
hour, then place on the slide dryer at 65-70 for another hour, then place in
the slide oven at 80 to melt - usually about 20 minutes. 

We have also tried microwave drying. 

We have also dipped the slides (after the tissue is on them) into
Poly-L-Lysine.

Even slides that are cut on Saturday, dry all weekend and not retrieved
until Monday morning are troublesome. 

Dottie - we happen to have a few boxes of Superfrost Plus slides here from
our Cytology department and are cutting some test slides as I type this.
Thanks for the suggestion! 

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Kim Merriam
Novartis
Cambridge, MA

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