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

[Kooijman, Esther]

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