[Histonet] cryosectioning - still bubbles between slide and section, spinal cord

[Christina Kreutzer]

Dear members,
first I wanna thank all the people who responded to my first email (Andrea,
Mesru and Emily).Your tips were very helpful. Unfortunately I am still
fighting agains bubbles/ wrinkles/bubble-shaped wrinkles between my slices
and the slide.

During the last couple of days I tried:

i) to adjust the temperature of the chamber and the sample, seems like
-16°C brings the best slices

ii) to cool the slide by putting it into the chamber and warming it with my
thumb just the second I held it towards the slice to adher

iii) to warm the slides by putting them on a 37°C warm heating-plate after
attachement

iv) to moisten the slides with a little bit of bidest before letting the
slices attach and put these slides on a 37°C heating plate
afterwards...that was kind of desperate try to straighten the slices slowely

v)  3 different intermedia before embedding the sample in pure Tissue Tek,
a) 30% Succrose, b) half 30% succrose and half Tissue Tek, c) half Tissue
Tek half PBS

but...nothing really helped. I still have wrinkles aka bubbles. Tomorrow I
am going the cut perfusion fixed rat spinal cord, hoping that this makes a
difference.

Anyway, I think I have some options left 1) trying to cut on a different
mikrotome and 2) Leica promised to send me some slide/embedding
solution/blade samples that worked for other groups.

Can anybody think of something I havent tried yet? Besides of raising slide
thickness and cutting for freefloating procedure?

I would be so thankful for any ideas/suggestions/tips!

Regards
Christina