RE: [Histonet] ovarian tissue coming off slides

From:"Hugh Luk"

Heather,

I believe your problem is not in your staining procedure, but in ONE of the 
following: tissue fixation, processing, or microtomy procedures.

I used to see problems like you described in tissues from Pacific Island 
locations that my hospital lab contracted with, where we had no control over 
the fixatives the tissues were shipped in (Large Igloo-type coolers stuffed 
with surgical specimens that had collection dates of "Months ago" AND we 
realized they were using some unbuffered, possibly undiluted formaldehyde 
solutions).

Please tell us more about your entire process.  My specific questions to you 
are:

1) Fixation proceedure; time and what chemical?  Is it alcoholic?
2) Tissue processor times for reagents?  How many tissue cassettes per run?
3) Temperature of waxes in tissue processor?
4) Temperature of wax in embedding center?  Do you keep the cassettes in 
warm paraffin in the embedding center?  If you do, how long before you embed 
them?
5) While embedding, do your tissue blocks look "Dry" or hard/brittle?
6) Microtomy (sectioning) thickness; 4-6 microns?  Do you "Soak" your tissue 
blocks after trimming them?
7) What type of knife/blades are you using?  Does it nicely ribbon off the 
knife?
8) What slides you are using, and vendor (I have a personal list of 
"Variable quality" slide vendors).
9) What water do you use for your water bath?  pH?  Do you add anything to 
the water?
10) Slide incubator, time AND temperature?  Is it a "Forced-Air" dryer?  How 
do the slides look when they are "Dry"?  Does water remain?  Is the paraffin 
slightly melted?
11) As for staining: Xylene purity, and is it "Recycled"?
12) Fish ovaries.  Decalcification?

In my experience; #10 is the most common pitfall.

I believe you should be able to provide stainable H&E slides with the 
tissues you have already processed, with just some "Tricks".  I have to 
admit though, I am a little worried about IHC or In-Situ proceedures.

Have you had successful staining?  What results you are seeing (clarity of 
cellular elements)?  I would think that your staining solutions are fine 
unless you have poor staining (as you mention changing your stains).

Take-heart in the fact that we, as a group (lab people doing histology), 
have seen this before and it has frustrated everyone to some degree.

Since my work location is only 10-15 minutes away from yours, feel free to 
call, or ask for advice, especially if you want me to look at something.  If 
you want me to try some microtomy and staining for you, I can do it at my 
location.  Maybe that will help with the trouble-shooting.

Hugh Luk, HTL (ASCP)
hluk@crch.hawaii.edu or hlkey@msn.com
Pathology Shared Resources Lab Manager
Cancer Research Center of Hawaii
1236 Lauhala Street, Room 316
Honolulu, HI  96813
Tel: (808) 440-5238
Fax: (808) 586-2982
Cell: (808) 226-4294

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>From: Heather A Leba 
>To: histonet@lists.utsouthwestern.edu
>Subject: [Histonet] ovarian tissue coming off slides
>Date: Wed, 07 Mar 2007 15:38:49 -1000
>
>After a month of trying to troubleshoot this issue can anyone offer some 
>suggestions? I'm mounting fish ovarian tissue and ooctyes and have 
>repeadedly had my sections fall off during the staining process (H & E) 
>during the second xylene step and also during the ETOH. I've tried using 
>tissue from different specimens, replacing my solutions during the 
>dehydration process, changing the staining solutions, using poly-L-lysine 
>coated slides, putting them on a slide warmer, letting them air dry 
>overnight, and now tried charged slides (but have not stained these yet). 
>I'm not sure what else to try and am at my wits end. Please help!
>
>Frustrated!
>Heather Leba
>
>UH Manoa, Zoology Dept.
>
>
>
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