Re: [Histonet] Intestinal Tissue Questions

From:Geoff McAuliffe

Hi Wendy: wrote:

>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
>are fixed.
>Fixation: 10% formalin
>Processing and embedding:  manual processing, paraffin embedding, 4-6
>micron sections
>Staining:  H&E staining
>Two questions:
>1.  After fixation, is it better to store my tissue (for ~1 month or so)
>in fixative, or in 70% ethanol?
How long are you fixing? 48 hours is a minimum, a week is probably 
better. Formalin works slowly. I don't know exactly what you are looking 
for but I would try to standardize my processing schedule so all tissues 
were treated for similar time periods. Storage in 70% is OK, but some 
cytoplasmic components may be extracted. I would just go ahead and embed 
rather than store in 70% for more than a week. Someone has certainly 
studied this, but back in the 1950's, or even earlier.

>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?
Smooth muscle (in the muscularis externa) does this. If you want nice 
cross sections cut the intestine open and pin it out flat on a piece of 
cork sheet (Home Depot) or dental baseplate wax (see your dentist, much 
cheaper than a supply house) using insect pins (#2 is a good size) while 
it is fixing. You can turn the cork/was sheet upside down in a jar of 
fixative. BETTER: don't cut it lengthwise, use a syringe or a pasteur 
pipet to wash the food, etc out of the lumen then tie off both ends with 
suture material/dental floss/thread leaving some fix in the lumen, 
enough to fill the gut but not stretch it out too much. Make a sausage, 
so to speak. After fixation, cut off the tied parts and you will have a 
nice, round piece of gut. Works better on small intestine than on large, 
more substantial wall in the small gut.
    Call me on the phone for a method to "jelly roll" the whole gut.

Geoff (who worked on the small intesting during the last century)

>Thanks for any advice.
>Wendy Bedale, Ph.D.
>Assistant Scientist
>Department of Biochemistry
>University of Wisconsin-Madison
>433 Babcock Dr.
>Madison, WI 53706
>608-262-3099 ext. 3266
>Histonet mailing list

Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029

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