Re: acetone fixation/frozen sections/spleen

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From:"Barry Rittman" <>
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thought that your comments were right on point.
One thing that most individuals do not consider is the composition of the
tissue. Tissue such as spleen which have a large proportion of cells with
relatively little fibrous components (except for capsule and trabeculae)
will always be more delicate to handle and often will need some extra care.
It is not uncommon for cells from spleen sinusoids to become detached and
settle elsewhere in the tissue. It may be that such tissues also do not
adhere as tenaciously to microscope slides because of the small amount of
ground substance and fibers present. In procesing to paraffin wax, these
tissues will usually show greater number of cracks and separation because
of the differential shrinkage. 		

At 11:57 AM 02/05/2000 -0700, you wrote:
>We use acetone fixation exclusively on murine, bovine, and rat spleen,
>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,
>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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