Author Topic: Dehydration before coverslipping?  (Read 4063 times)

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Offline Isobel

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Dehydration before coverslipping?
« on: October 20, 2008, 09:44:06 AM »
After staining I usually coverslip the sections using some sort of permanent mounting medium. Should I dehydrate the sections with ethanol series + xylene before coverslipping? Up to now I have only air dried the sections before coverslipping (after HRP/DAB staining + final rinse with dH2O), and this has seemed to work just fine. What are the benefits of the ethanol/xylene dehydration?

Dehydration before coverslipping?
« on: October 20, 2008, 09:44:06 AM »

Offline excalibur

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Re: Dehydration before coverslipping?
« Reply #1 on: October 21, 2008, 12:06:27 PM »
Allowing sections to air dry can cause poor morphology.
Paula K. Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
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Offline ImmunoNYC

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Re: Dehydration before coverslipping?
« Reply #2 on: October 31, 2008, 08:44:34 PM »
I agree with Paula. Plus dehydrating ensures there is no water left before coverslipping with organic mountant. If you have residual water you can artifacts from the water. Also your slides are so much prettier after the organic "washes" ... No more pan pen, dirty slides etc ...

Offline Isobel

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Re: Dehydration before coverslipping?
« Reply #3 on: November 01, 2008, 07:25:50 AM »
Thanks, I will do the dehydration in the future.

Just an additional question. excalibur says air drying will cause poor morphology. I let my sections air dry at room temperature for a while after sectioning. Will this also affect the morphology?

Offline excalibur

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Re: Dehydration before coverslipping?
« Reply #4 on: November 01, 2008, 04:42:43 PM »
No, because they are still in the paraffin. The water must further be removed by heating in the oven.
Paula K. Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
8901 S Santa Fe, Suite G
Oklahoma City, OK 73139
405-759-3959
www.excaliburpathology.com

Offline Isobel

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Re: Dehydration before coverslipping?
« Reply #5 on: November 02, 2008, 04:17:42 AM »
As they are (pre-fixed) freeze sections, they are not in paraffin.

Offline gula

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Re: Dehydration before coverslipping?
« Reply #6 on: November 02, 2008, 04:46:06 AM »
I think in this case, airdrying will help to a better adhearence of the slides. Without this, I fear, the slides would go off. And this is worse than bad morphology.
In my opinion (without practical experience with prefixed frozens) I assume, that airdrying of properly fixed specimen doesn't matter, because morphology of frozens is always worse than that of the paraffin-slides.
After the IHC-procedure of FFPE-sections it should make a difference, because the tissue was heated and treated and is then more sensitive for drying-artefacts. - Just theoretical thoughts.
gula

Offline Isobel

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Re: Dehydration before coverslipping?
« Reply #7 on: November 02, 2008, 06:46:30 AM »
Ok. Thanks again :)

Offline Cardio

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Re: Dehydration before coverslipping?
« Reply #8 on: November 10, 2008, 01:04:30 PM »
Isobel,

I would suggest for you to try both ways. I do both depending on what is needed. If publication quality slides are needed I would do a wet mount and get the pics. For long term storage you can either dehydrate air or ethonal/xylene. If your tissue is semi-fixed in PFA be carefull of the ethanol. It can make the tissue shrink.

Let me know if you see a difference.

Re: Dehydration before coverslipping?
« Reply #8 on: November 10, 2008, 01:04:30 PM »