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Fixation and embedding of fetal mouse ovary

immunohistochemistry fixation embedding histology

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#1 nofear

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Posted 28 September 2012 - 01:54 PM

Hi All,
I work with mouse ovaries at several stages of development. I am having serious trouble in processing (fixation and/or embedding into paraffin) fetal ovaries: when I cut the paraffin with a microtome (5um thickness) the sections have one or more of the following problems: 1) the sections have several folds along its lenght (appear wrinkled) 2) the ovary seems 'broken' with several empty regions inside the tisue (like if it expanded from the inside) 3) after placing the paraffin strip in the waterbath, the tissues in the middle of every section 'detach' from the paraffin (like if the entire section is expanding), at this point after placing the strip onto a slide the tissues usually have either or both problems 1) and 2). I need to point out that I don't have any problem whatsoever when processing newborn or post-birth ovaries. I can't figure out where the problem is because sometimes everything goes fine (rarely though), sometimes the above mentioned problems are small or mild, other times it's just a disaster. This great variability just drives me insane.

The following protocol for fixation and embedding works totally fine for ovaries at any stage except embryo:
-fixation in 4%PFA/PBS overnight (for embryo ovaries I tried also from 1h to several days... no difference in fixing the problems)
-30% EtOH (10 min x 3)
-50% EtOH (10min x 3)
-70% EtOH (10 min x 3)
-90% EtOH (10 min x 3)
-100% EtOH (20 min x 4)
-Toluene (20 min + 10 min x 2)
-paraffin

Any insight would be really appreciated!
Dan

#2 bob1

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Posted 28 September 2012 - 02:45 PM

It sounds like a cutting problem - try cooling the block before cutting, make sure the knife is new or recently sharpened too.





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