[Histonet] Re: How to get good cross sections of the mouse small intestine

[Gayle Callis]

Jian,

You need to rinse the feces out of the lumen.  Remove the small intestine 
INTACT but carefully by lifting it out of mouse.  You can detach the 
mesentary as you do this but do not rip the sample out, you do NOT want to 
tear holes in the intestine.   Fill a 50 ml syringe with PBS, and use a 
dulled 18 guage needle - insert into one end of the intestine (easier from 
the ilium side) and inject the PBS.  After rinsing out feces, use a syringe 
filled with neutral buffered formalin, and inject that so the lumen 
distends.  This will fix the villi much faster and get rid of digestive 
enzymes and left over fecal matter.

You can clamp off the ends of the intestine or have sutures ready to tie 
off ends but you need to do this as the fixative is flowing out the 
opposite end of the intestine - overdistending the gut may not be a good 
idea, you need just enough to fix but not create a huge sausage like 
structure.  Someone helping is nice, as they can tie off one end while the 
needle is removed to tie off the other end or use mosquito hemostats and 
clamp off.   The idea is to fix the inside of intestine quickly to preserve 
villi.   The gut can be stretched out in distended state or you can drop it 
into fixative then cut cross sections from each individual area of 
interest, process and embed on end.  WE do this but NOT for cross sections, 
but mid sagittal sections however the intestine is well fixed, and with 
clean results.

After fixation, you can store in 70% alcohol until processing.  Just make 
sure you have total fixation or the 70% will finish it off for you.  We are 
careful to not over process these tissues, they are small and delicate, and 
use a shorter processing schedule than for larger tissues.

   At 02:11 PM 1/23/2006, you wrote:
>Dear all,
>
>I am a new histoneter.  I am just starting to do some mouse small
>intestine histopathology and wondering whether anyone can provide a
>method (protocol) for how to fix and embed mouse small intestine to
>generate good cross (paraffin) sections with as many intact villi and
>crypts possible for H&E and IHC   Particularly, I need to evaluate at
>least 5-10 different area of the small intestine to score the
>regeneration of crypts and the extend of apoptosis at various time point
>after irradiation.
>
>I have used neutral buffered formalin as fixative and manually bundle
>four pieces of 0.5 cm-long tubes together before embedding at a clinical
>pathology lab.  The sections looked OK, but many appear to have a lot of
>floating villi in the lumen and limited number of crypts extending into
>to the villi connected to them.   Since we can not always get our
>tissues processed after O/N fixation, we sometimes keep the tissues in
>NBF for several weeks.  Is this too long, or should we transfer them to
>70% ethanol?

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)