Cracks in paraffin sections
Can somebody tell me how to get rid of cracks in paraffin sections?
When I prepare paraffin sections (7µm) 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 45°C) 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 4°C.
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 58°C) 1:1 at 45°C
over night n-butyl acetate / paraffin 1:4 at 52°C
3x2h paraffin changes
embedding in freshly molten paraplast
solidifying at a -5°C 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 (45°C) and are left over night in a 37°C
incubator for further drying.
Any suggestions for solving this problem are highly appreciated!
Thanx in advance for help
Max-Planck-Forschungsstelle für Ornithologie
<< Previous Message | Next Message >>