[Histonet] autoradiography frozen human brain sections fall off, white matter issue, solved !!!

[Kooijman, Esther]

Dear Histonetters,

Thank you Barbara, Wendy and Brett and others that were so kind to give me hints and tips !

I managed to solve the problem, all tissue stays on. 
I have used Superfrost Plus Gold. And a absorbent pouch inside the storage case. After cutting keeping them melting on longer about an hour at room temp. Then again like we used to in silica in the fridge ON. When getting them out leaving them in the box for an hour to defrost. And........ all the tissue stays on during the complete procedure !!! @Barbara no need to try the fixing step ;-)

Thank you all for your input !

Have a nice weekend. 

Esther

-----Oorspronkelijk bericht-----
Van: Connolly, Brett M [mailto:brett_connolly at merck.com] 
Verzonden: vrijdag 2 oktober 2015 16:08
Aan: Kooijman, Esther
CC: histonet at lists.utsouthwestern.edu
Onderwerp: RE: [Histonet] autoradiography frozen human brain sections fall off, white matter issue

Hi Esther,

We do a lot of autoradiography on rodent, primate and human brains sections. Our protocol is similar to yours except we do not dry the slides after incubating in the buffer and before adding the radiotracer.

The experiment is performed with the slides in a rack and totally immersed in the solutions in staining dishes.

1. incubate slides in assay buffer 15 min.  at RT 2. add radiotracer to buffer, gently mix and incubate 90-120 min. at RT 3. wash  GENTLY (no agitation) in ice-cold wash buffer 3 x 3 min. We set up 3 containers and transfer the slides.
4. wash in ice-cold DI water 5 seconds.
5. air dry

The Superfrost gold slides are supposed to improve adherence and would be worth a try. It could be your other Superfrost slides are old or a bad batch...which I think has been experienced by some people on the list.

We do store our sectioned slides at -70C until use and then bring them up to RT the day of the experiment. They are sectioned, dried at RT for 15-20 min, and then store at -70C... No drying in the fridge.

Good luck,
Brett


Brett M. Connolly, Ph.D.
Principle Scientist,
Translational Biomarkers - Imaging
Merck & Co., Inc.
PO Box 4, WP-44K
West Point, PA 19486
brett_connolly at merck.com
T- 215-652-2501
F- 215-993-6803

-----Original Message-----
From: Kooijman, Esther via Histonet [mailto:histonet at lists.utsouthwestern.edu]
Sent: Friday, October 02, 2015 5:20 AM
To: histonet at lists.utsouthwestern.edu
Subject: [Histonet] autoradiography frozen human brain sections fall off, white matter issue
Importance: High

Dear Histonetters,

Does anyone of you have experience in autoradiography and could help me with the problem falling of tissue  ? Or suggestions ?

We have problems with falling off sections of human brain tissue.
We have done the same experiments and cutting in rodent tissue without any problems. Our protocol for autoradiography  :
Snap frozen tissue, cut in cryo 20um sections on Superfrost plus glass, drying ON in the fridge on silica.
Stored in -20.
Day of the experiment.
Getting them on room temperature about 45 minutes, washing/dehydrate on room temperature in 5mM Tris (HCL PH7.4) buffer, 20 minutes.
About  30 minutes - an hour drying at room temperature.
Then incubation with the radio tracer for 30 minutes, room temperature, just dripping the 1 mL solution to completely cover the superfrost plus glass and ditto tissue.
Turning the glass to get the solution of the glass,  dipping in ice cold tris washing buffer 5mM (HCL PH7.4) 1.5 minutes and here the disaster starts with falling off extensive white matter falling off...... its washing steps of only 1.5 minute.

What can I do to prevent this, any idea ? I am out of clues. I have tried to dry the sections longer after cutting sections, I have tried to pre wash in either room temp or cold buffer. Hope someone can help me.
Would Superfrost plus gold glass be better ?

Kind Regards,

Esther Kooijman
Research Technician
VU University Medical Center
Nuclear Medicine & PET Research
Email: e.kooijman at vumc.nl<mailto:a.metaxas at vumc.nl>
Amsterdam
The Netherlands

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