acetone fixation/frozen sections/spleen

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From:Gayle Callis <>

We use acetone fixation exclusively on murine, bovine, and rat spleen, without
any loss of sections or bubbling problems.  Our morphology is excellent.
Immunostaining is good.  Have found that hydrogen peroxide/methanol blockers 
chews up sections, use Dako's peroxidase block, but the methanol/H2O2
blocks are not recommended for CD marker staining anyway (per PharminGen and
even Biogenex).  

We are careful to air dry the sections a minimum of 30 min, up to 4 hours, and
do cold 4C acetone fixation.  Stain or store, but storage at -80C then bring to
RT, the container should be kept sealed until slides equilibrate to RT, the
condensation formed by immediate opening of slide box can cause crummy
morphology also.  

You did not say how cold your cryostat was for sectioning, warming
to -16 to -17C gave us a section without laddering, shattering, and very flat
mounted on a Plus Charge slide.  Could it be your section loss is a tidge
related to not getting a flat section.  Also when you rinse the section, do you
allow the buffer to flow from the top of slide, gently over the section.  I
messed up a bunch that way.  

Snap freezing correctly avoids freezing artefact, than creates a spleen with
separation gaps in section, freezing was too slow.  

I don't know if this helps, sure a load of blab though

Gayle Callis 

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