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Author Topic: Peeling off, destruction,etc.  (Read 5420 times)

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Offline Fredi

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Peeling off, destruction,etc.
« on: August 20, 2005, 05:11:08 AM »
Hi,

we've got a serious problem with sections peeling off during HIER and even when they stick on the slide they are almost always so destructed that it is impossible to evaluate them. We have tendon, ligament and cartilage as tissue. We used Superfrost Plus, which was absolutely hopeless, we tried Poly-L-Lysin coated slides which are a lot better, we tried Poly-L-Lysin/Ponal, Poly-L-Lysin/Pritt, all not better than Poly-L itself. But still the architecture is destructed. We are down to not use HIER, but for some of our ab it won't be possible. For HIER we used pH9 Dako Target Retrieval Solution or pH6 Citrate Puffer, 95-98C in water bath for 20 min. pH9 was of course worse. We checked on fixation and so on and that should be OK.
I haven't heard about the gelatin in waterbath yet so I will try that, too. And this coating with gelatin. But will that not wash off in the heat, too?
What drying regime is preferrable? Don't I ruin my antigens (espec. collagens) when drying for longer in 56C oven?
How sensible is it to try a formic acid retrieval, if the manufactorer suggests pH9 ?
We are quite desperate at the moment and are very grateful for every kind of advice.

Fredi

Peeling off, destruction,etc.
« on: August 20, 2005, 05:11:08 AM »

Offline excalibur

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Peeling off, destruction,etc.
« Reply #1 on: August 21, 2005, 09:32:03 AM »
Are your sections frozen or paraffin?

If paraffin, my best guess is your slides are not completely dry before deparaffinization. After you pick up the sections onto the slide from the water bath, allow them to air dry an hour or two. Then you can place them in a 58 C oven for no less than 30 minutes. If they have airdried first, even leaving them in the oven overnight should not have adverse effects.

Superfrost Plus slides should be the best choice. You may have received a bad lot. This has happened to me.

Make sure when using the coated slides the water bath is filled with distilled water and not tapwater.

Gelatin in the waterbath is the ancient method we used, and still do for routine H&Es, before silane and poly-l-lysine coated slides. It will not hold up for HIER. Even gelatin-chrome alum is not as good as the coated slides.

Good luck
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline Fredi

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Peeling off, destruction,etc.
« Reply #2 on: August 22, 2005, 03:43:39 PM »
Thank you That's really helpful. We do have paraffin section. Is there anything I can do about the tissue architecture? Guess we really just have to try to avoid HIER. Hope it will work.

Thanks again!

Fredi

Offline excalibur

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Peeling off, destruction,etc.
« Reply #3 on: August 22, 2005, 04:04:35 PM »
If you tissue is fixed, dehydrated, cleared, and infiltrated with paraffin correctly before embedding and sectioning you should have no problems with HIER destroying morphology unless you overdo it.

The first rule of histology is fixation, fixation, fixation.  :wink:
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Peeling off, destruction,etc.
« Reply #3 on: August 22, 2005, 04:04:35 PM »