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As anyone found a way to make this work?

Need some advice!
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Offline julied

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Problem with heat retrieval and tissues falling off
« on: August 28, 2007, 04:06:50 PM »
Hello,
I am having problems with skin tissue sections falling off slides after heat retrieval using sodium citrate buffer pH 6.0 (30 min at 95-96C)
I read some of the entries dated 2005 and want to know if anyone as come up with anything that actually works.  
Julie

Problem with heat retrieval and tissues falling off
« on: August 28, 2007, 04:06:50 PM »

Offline dutchmaninvienna

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Re: Problem with heat retrieval and tissues falling off
« Reply #1 on: August 29, 2007, 02:38:03 AM »
You need to use coated slides, normally silane coated (charged) slides should be OK for 30 min citrate treatment. If the silane coated slides (superfrost) do not work you should try superfrost plus slides (also charged, but somehow stronger). We use these for 60-90 minutes HIER with EDTA pH 8.5-9. If this problem appeared suddenly you should find out if you have a non-working batch of slides. Another possibility might be the water. We found out that, for some kind of reason, if we make the EDTA buffer in deionized water, the sections tended to fall of. When we use distilled water this problem appears only seldom, for instance with very old overfixed archival material.

hope this answers your question.
jan
« Last Edit: August 29, 2007, 02:52:26 AM by dutchmaninvienna »
Jan Bauer, PhD
Associate Professor
Div. of Neuroimmunology
Center for Brain Research
Medical University of Vienna, Austria

Offline excalibur

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Re: Problem with heat retrieval and tissues falling off
« Reply #2 on: August 29, 2007, 01:18:02 PM »
Totally agree with Jan and will add that the tissue sections should totally airdry and then be placed in a 60C oven to continue drying for at least an hour, ( can be longer, even overnight).

Water trapped under the section that has not completely dried is the main cause of tissue falling off during any staining procedure.
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 julied

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Re: Problem with heat retrieval and tissues falling off
« Reply #3 on: August 30, 2007, 03:07:31 PM »
Thank you very much to you both. Will try the distilled water in place of deionized.
Superfrost slides are already being used.

Julie

Offline dutchmaninvienna

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Re: Problem with heat retrieval and tissues falling off
« Reply #4 on: August 31, 2007, 10:05:00 AM »
@julied

Hope it works. Let us know if it does!
Jan Bauer, PhD
Associate Professor
Div. of Neuroimmunology
Center for Brain Research
Medical University of Vienna, Austria

Offline ydnew

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Re: Problem with heat retrieval and tissues falling off
« Reply #5 on: December 17, 2008, 03:30:19 PM »
I have also had the problem of tissues (especially human normal and cancerous breast) falling off the slides after heat induced antigen retrieval (5 min in Sodium Citrate buffer, pH 6.0, in pressure cooker)  even though the tissues are mounted on superfrost plus slides. I am going to try switching the water base of the buffer to distilled water instead of deionized as suggested, but had also read that a 10 minute post-fixation of slides in 10% neutral buffered formalin after deparaffinization can help. Has anyone tried this or heard of this?

Thanks!

Offline Cardio

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Re: Problem with heat retrieval and tissues falling off
« Reply #6 on: December 17, 2008, 04:56:54 PM »
This is from the knowledge section of IHCWorld.

Why my section falling (coming) off slide?
 How to prevent section from falling off slide?
 
Insufficient fixation or unfixed tissues tend to come off slides more easily.
 Fix tissues longer to get completely fixation of tissues. 
Formaldehyde fixed frozen sections are more prone to falling off slides.

 
 Try to dry slides for much longer time or use alternative fixation such as acetone or alcohol.  

Tissue sections tend to come off slides more often on regular slides (uncharged or uncoated).

 
 Always use positively charged or coated slides for immunostaining.
You may just have had a bad batch of slides.

 
 Replace with a new batch of slides.
 
 
Hand lotion or cream contaminating the waterbath.  Avoid hand lotion or cream contaminating waterbath by wearing gloves.
The disposable blades have oil on them.

 
 Clean disposable blades with xylene.
 
Wrinkles presented in the sections during the initial mounting may contribute to problem for falling off sections.

 
 Try to spread the sections out and mount the sections on slides with wrinkle free.
 
Paraffin sections may not be dried completely before placing in the oven.
 Allow paraffin sections to air dry at least 30 minutes before placing in the oven at 56 C overnight.

 
 
Frozen sections may not be dried completely before fixation and immunostaining procedure. Allow frozen sections to air dry for at least 30 minutes before fixation and then air dry for another 30 minutes before immunostaining.
Antigen retrieval procedure can cause sections to come off slides, especially when EDTA (pH8.0) or Tris-EDTA (pH9.0) such high pH antigen retrieval solution is used.

 
 Use low pH solution such as citrate buffer (pH6.0) antigen retrieval Solution to replace EDTA (pH8.0) or Tris-EDTA (pH9.0) retrieval solution if it is possible.
 
Distilled water alone may make sections come off slides easy.
 Always use buffer solution to wash or rinse slides.

 
 
Bone (especially the cartilage) tends to fall off slides after HIER.

 
 Try other alternative retrieval methods such as enzyme digestion.
 
Antigen retrieval devices may be trouble.

 
 Try to use waterbath or steamer in stead of microwave or pressure cooker.

Plain water in waterbath for mounting paraffin sections.
 Add some gelatin in waterbath for mounting paraffin sections.

 
 
If you have tried everything above and the problem persists.
 Try gelatin coated slides and it has been working fairly well.
 
« Last Edit: December 17, 2008, 04:58:29 PM by Cardio »

Re: Problem with heat retrieval and tissues falling off
« Reply #6 on: December 17, 2008, 04:56:54 PM »