murine gut frozens

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From:Gayle Callis <>
Date:Tue, 6 Jul 1999 15:28:41 -0600

The mouse intestine is washed with PBS, to rinse out fecal matter.  Mousie doo
does not section well  (no further derogatory comments about mouse
poop!) and has a tendency to roughen up the villi.  After washing the
gut gently (use a large bore needle cut off and dulled to get rid of
sharp hypodermic tip/cut edges) fill intestine with OCT:PBS mixture
1:2.  This extends gut, fills gaps around the villi and permits you
to see peyers patches on the surface easily.  One becomes experienced on
finding duodenum, jejunum and ilium (sp?).  Cut filled gut into pieces
with razor blade, put pieces in plastic tissue tek disposable mold,
even a concentric circle arrangement, a bit leaks out, work quickly
add undiluted OCT around the gut, snap freeze in dry ice/isopentane slurry 
via immersion. Takes a bit of practice. 

Make sure the outside of gut stays moist with PBS, and do not use too
much PBS when mixing with OCT, excess PBS makes a bad matrix for
sectioning (excess water and similar sectioning as poop, shattered).  
Section at  -16 to -20C, pick up on plus charge slides, air
dry up to 4 hours, 30 min is minimum.  Fix with 4C acetone 10 min, air dry
15 min, fix again 10 min in 4C acetone, air dry 30 min, stain.  Do not use
a strong peroxidase blocker (DAKO has a 0.03% hydrogen peroxide) or make
up same concentration in PBS, 0.05% works well also. Those bubbles create
havoc with delicate frozens, or an overrinser, gently flow PBS from above
section avoids forcible mechanical manipulation of section. 

When dissecting gut, be sure to not poke holes in gut, or leave a lot of
fascia/fat (interfers with sectioning) on surface. This is routine 
small intestine freezing protocol for us, villi remain intact, undamaged.
Stay in touch if this works for you.

Gayle Callis 

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