Paraffin sectioning for in situ

Vasundhara Kandachar kandachar at
Tue Jul 10 18:32:05 EST 2001

Hi all!

We are trying to prepare 10 micron sections of spleen,
thymus and lymph nodes from 6-8 week old mouse for in
situ hybridization (to mRNA).  

We have encountered two major (related?) problems:

First, we've found long rectangular holes in the
sections, as if a piece popped out while sectioning.  

Second, sometimes the entire section breaks into
shards leaving only the parafin ribbon without tissue.
 We have been told that perhaps these 
sectioning difficulties are due to dehydration or
parafin infiltration problems. 

Our procedure (below) has been taken from established
protocols for sectioning animal tissue.  
We process the tissue as follows:
Fixation: 4% paraformaldehyde in PBS overnight (RT).
Wash:  0.1 M Sodium phosphate buffer at 4 degrees C
Dehydration:  5 minutes in each of six different
concentrations of ethanol at RT (increasing from 10% ,
25%, 50%, 70%, 95%, and 100%).
Replaced ethanol:  Used 50% xylene in 100% ethanol,
then 100% xylene (both for 5 minutes at RT.  Paraffin
Infiltration: added 2 pellets of parafin every 10
minutes at 60 degrees C for 8 hours and then resumed
adding pellets the next day for another 8 hours.
Evaporate the xylene:  Left the vials without caps at
60 degrees C overnight.
Replace with 100% parafin:  twice per day for 10 days.
Pour parafin with tissue into molds and left 4 degrees
Cutting sections:  Used hand operated microtome with
freshly sharpened steel knife to cut 10 micron
sections from blocks that have been trimmed and 
cooled on ice.

Please help!  What are we doing wrong???
(the person cutting the sections is a very experienced
plant histologist who uses the same microtome
successfully for preparations of thin sections for 

Thanks in advance.

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