[Histonet] mouse intestine fixation (long description, two ways)
We have done this two ways:
1) Never open the intestine. Remove whole intestine carefully to prevent
holes which leak during rinses. For easier handling of whole intestinal
tract, we remove large intestine from small intestine at cecal
junction- easier to rinse two pieces. It takes a bit of practice to see
where you can start the rinse. In general, we start rinse at narrow opening
and rinse toward larger opening.
Rinse fecal contents out with PBS - 50 mls, it may take a bit more. Use a
50 ml syringe with a #18 guage needle (dulled so as to not damage internal
mucosa) for easy insertion into open end of intestine. Be gentle, do go
int too far or tear tissue. After rinse, fill intestine with NBF until it
runs out, it should appear sausage like (sorry folks! for the
analogy). Immerse NBF filled intestine into 1:20 volume tissue to NBF for
desired fixation time. After fixation, slice intestine into lengths. We
found NBF filled intestine stays distended by fixative, and prevents lumen
from collapsing on itself. This allows for immediate, good fixation of
delicate villi damaged by residual gut digestive enzymes.
We maintain the "tube", so to speak, in order to not have the problem you
are experiencing. The processed lengths fill nicely with paraffin, and
remain as a tubular structure. We embed for midsagittal or longitudinal
orientation to visualize PP on serosal on outside (serosal surface) and
villi on internal mucosal surface.
2) Be sure you open gut on midline away from peritoneal fascia
attachments. This can be done with a tiny straight dissection scissors,
called iris scissors, or a very sharp, fine tipped cuticle scissors from
WalMart. Lay your opened strips on lengths of filter paper (buy large
sheets, cut into strips, get smooth paper) , wet strips with PBS before
putting unfixed tissue on paper, prevents sticking. You can manipulate gut
a bit so it sticks, with villi facing up, then fold paperstrip like an
accordian, immerse into NBF. If you work in a hood, then wet the paper
with NBF, the serosal surface will fix a bit, and you won't leave gut
sticking to paper when you embed. The fixed intestine will be perfectly
flat. Process folded paper with gut inside a cassette, process but remove
paper gently at embedding.
If all you do is H&E, you can store in NBF.
>Hi to all of you,
>I'm quite new to histology and have a few basic questions.
>Here's what I'm doing:
>Tissue: mouse intestine; small and large intestine is harvested, cut
>open longitudinally, and rinsed with PBS prior to fixation; 3 cm lengths
>Fixation: 10% formalin
>Processing and embedding: manual processing, paraffin embedding, 4-6
>Staining: H&E staining
>1. After fixation, is it better to store my tissue (for ~1 month or so)
>in fixative, or in 70% ethanol?
>2. I'm attempting to get transverse sections of the intestine; sometimes
>I notice that my tissue ends up inside out (mucosa facing outward). I
>think it may happen when I use a razor blade to cut the tissue
>longitudinally vs. using scissors to make the longitudinal incision, and
>am going to test that. Has anyone had any experience with this?
>Thanks for any advice.
>Wendy Bedale, Ph.D.
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)
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