Cracks in paraffin sections

From:Sylvia Kuhn


Can somebody tell me how to get rid of cracks in paraffin sections?

When I prepare paraffin sections (7m) of whole sparrow brains (roughly 
17x15x9mm) they show after staining lots of cracks that are spread all over 
the section. Sometimes they are even that big and numerous that they 
literally disrupt the section into small shreds when they float in the warm 
waterbath (37 to 45C) prior to mounting.

Does anybody know what I did wrong?

The tissue got preliminarily fixed by perfusion of the whole bird with 
Sigma fomalin solution (10% Formalin (approx. 4% formaldehyde), Neutral 
buffered solution). After dissection of the brain it was postfixed over 
night in the same solution at 4C.

I tried already several embedding protocols, the one that I use now is one 
of the shortest:

4x1h PBS buffer pH7.4
2h 30% ethanol
1h 50% ethanol
1h 70% ethanol
over night 80% ethanol

1h 90% ethanol
1h 96% ethanol
3x2h 100% ethanol (dehydrated over a molecular sieve)
2x1h methyl benzoate
over night methyl benzoate

3x2h n-butyl acetate
3h n-butyl acetate / paraffin (paraplast, melting point 58C) 1:1 at 45C
over night n-butyl acetate / paraffin 1:4 at 52C

3x2h paraffin changes
embedding in freshly molten paraplast
solidifying at a -5C cold plate
leaving the block over night in the fridge

I cut the sections on a rotary microtome with disposable blades using every 
now and then another part of the knife.

The sections are mounted in a warm water bath where they flatten and 
enlarge to the original size of the brain prior to dehydration (the brain 
width changes from approx. 17mm in the formalin solution to approx. 13mm in 
the 100% ethanol and back to approx. 16mm on the slide). The slides dry 
several hours on a heating plate (45C) and are left over night in a 37C 
incubator for further drying.

Any suggestions for solving this problem are highly appreciated!

Thanx in advance for help

Sincerely

Sylvia Kuhn


Max-Planck-Forschungsstelle fr Ornithologie
Arbeitsgruppe Fidler
Seewiesen
Postfach 1564
D-82305 Starnberg-Seewiesen
Germany





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