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Author Topic: Processing WAT for H&E  (Read 4658 times)

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Offline RendinaOSU

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Processing WAT for H&E
« on: August 27, 2010, 12:03:01 PM »
Hello,

We have been trying to perform H&E on white adipose tissue, however we are having some issues.  We process all of our other tissues (i.e., liver, lung, heart, aorta, spleen) using the method below after we remove the tissues from 10% NBF:

Formalin 2 min
Alcohol 70% 20 min
Alcohol 80% 20 min
Alcohol 95% 20 min
Alcohol 95% 20 min
Alcohol 100% 20 min
Alcohol 100% 20 min
Alcohol 100% 20 min
Toluene 30 min
Toluene 30 min
Wax 50 min
Wax 60 min

However when we use this protocol with WAT, our tissues never seem to be completely dehydrated, therefore when we try to cut it we cannot.  As mentioned before our tissues have already been fixed and stored in 10% NBF, so we really dont have an option there, but is there any other advice you could give as to optimizing the processing procedure for such a tissue?

Thanks!
BR

Processing WAT for H&E
« on: August 27, 2010, 12:03:01 PM »

Offline Rui

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Re: Processing WAT for H&E
« Reply #1 on: August 27, 2010, 03:24:37 PM »
How long are you leaving the specimens in 10% NBF. Fatty tissue takes much longer to fix and you should account for this with added fiaxtion times. I would try increasing the in process Formalin fixation to 45min-60min.

Also, you may want to create a protocol just for WAT with longer step times as faty tissue tends to take longer to infiltrate properly during VIP. Adding ten minutes to each alcohol step may help.

Offline RendinaOSU

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Re: Processing WAT for H&E
« Reply #2 on: August 27, 2010, 04:22:32 PM »
Thank You!

The only problem is that they are initially in the 10% NBF for quite a long period of time (we just started developing a protocol and processing our tissues from prvious studies).  So in theory they've been in formalin for a couple of monthes and we are just now started to put them in blocks.  We could try increasing the times during the processing, thanks.  You wouldn't happen to have a protocol you've used before for WAT?

Thanks
BR

Offline excalibur

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Re: Processing WAT for H&E
« Reply #3 on: August 28, 2010, 08:41:25 PM »
Adipose tissue is fat. So you need to dissolve out the fat to get a good infiltration of the paraffin.

Since your tissue is fixed sufficiently, I suggest the following program because your times are too short and you also need to dehydrate better before using the solvent, I suggest using xylene, that will dissolve the lipids.

I am assuming formalin is in the first position on the processor which is why you have the 2 minute step. With a new program for your adipose tissue it can be skipped altogether or leave the short time in it if you do not know how to skip a station. Usually just setting the time to zero will cause the station to be skipped.

The 80% and one of the 95% can be removed from the machine and this will also help to better process your other tissues.

Try this with vacuum at every station:
70% 45 min
95% 45 min
3 100% 45 min each
3 xylene 45 min each
3-4 paraffins 1 hour each

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

Offline RendinaOSU

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Re: Processing WAT for H&E
« Reply #4 on: August 30, 2010, 11:37:27 AM »
Thank you so much!  We will try this!  Quick question, is the vacuum component of this procedure absolutely Necessary?  I do not believe we typically use this so I'm not even sure our  processor has these capabilities.

Offline excalibur

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Re: Processing WAT for H&E
« Reply #5 on: August 31, 2010, 10:26:11 AM »
It is not absolutely necessary, but it does help. Processors made prior to the 1980s and some still today did not have heat or vacuum.
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Re: Processing WAT for H&E
« Reply #5 on: August 31, 2010, 10:26:11 AM »