[Histonet] thick paraffin sections for stereology

[Mollie Lange]

Hello Everyone,
 
Help!  I need some fixation and processing alternatives. 
 
We need to cut 20-30 micron thick paraffin sections of post-natal day 7-14 mouse brains.  Immunocytochemistry for apoptosis inducing factor (AIF) will  be performed and then the positive cells will be counted using unbiased stereology (optical fractionator).  
 
Fixation is 4% paraformaldehyde perfusion with a 4-6hr post-fixation.  We dehydrate in graded ethanols, clear in cedarwood oil and methyl salicylate and embed in paraffin.  Processing steps are as short as possible to ensure adequate dehydration, clearing and infiltration. All steps are done by hand.  This protocol is okay for 6-10 micron sections, but not for the required thicker sections. The tissue just completely shreds.  Soaking the block face does not give good results.
 
Methacarn fixation is good for cutting thicker sections, but unfortunately it is not compatible with ICC for AIF.
 
Are there other fixation, processing or sectioning procedures we should try?  Bouin's?  Methanol?  Immersion rather than perfusion?  
 
Any and all insights are most appreciated.
 
Thanks,
Mollie Lange
International Center for Spinal Cord Injury
Kennedy Krieger Institute
Baltimore, MD 21205



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