[Histonet] Colon whole mounts and counting aberrant crypt foci (ACF)

From:"John McGinley"


Our lab is just beginning to study colon cancer using a rat model and I was
wondering if anyone out there has experience with making colon whole mounts
and counting aberrant crypt foci (ACF). It looks like the most accepted
method in the literature is cut the colon along the longitudinal axis, prep
as a whole mount (mucosal side up), wash in PBS, fix in 10% formalin
overnight, rinse, stain with methylene blue and look for ACF at x40. 

One of the problems I've been having is getting the whole mount to lay flat
and adhering to the slide during fixation. I've been blotting the serosal
layer with kimwipes to remove excess fluid prior to making the whole mount
on plain glass slides. Some reports in the literature indicate that the
colon is mounted or pinned to a cork board during fixation to help it lay
flat, stain while free floating and then transfer to a slide for microscopic
evaluation. This seems like a lot of work and I would rather mount the
tissue to a slide once and be done with it. Should I try using silane
treated or poly-lysine slides instead? 

When looking for ACF under the microscope should the whole mount be cover
slipped using aqueous mounting media or just left wet? 

What is the best method for counting the number of ACF? Should I use a
reticle, a cm grid transparency overlay on the tissue, etc.?  Any
information you can provide would be appreciated. Thanks in advance.



John N. McGinley, HTL(ASCP)
Cancer Prevention Lab
Colorado State University
111 Shepardson bldg. 
1173 Campus Delivery
Fort Collins, CO 80523-1173
Ph: (970) 491-3041
Fx: (970) 491-3542
Email: john.mcginley@colostate.edu 
Web: www.cpl.colostate.edu

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