[Histonet] thick paraffin sections for stereology

Mollie Lange lange <@t> kennedykrieger.org
Thu Oct 18 13:41:34 CDT 2007


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



Disclaimer:
The materials in this e-mail are private and may contain Protected Health Information. Please note that e-mail is not necessarily confidential or secure. Your use of e-mail constitutes your acknowledgment of these confidentiality and security limitations. If you are not the intended recipient, be advised that any unauthorized use, disclosure, copying, distribution, or the taking of any action in reliance on the contents of this information is strictly prohibited. If you have received this e-mail in error, please immediately notify the sender via telephone or return e-mail.


More information about the Histonet mailing list