Mouse intestine cryosection problem

From:Gayle Callis

I rinse all fecal matter out of intestine with PBS then fill with a 1:1
diluted OCT ( with PBS). Mouse poop tends to create crunchy sections, torn
and not very flat. This distends gut to see lumen/Peyers patches on serosal
surface.  Intestine can be cut into sections, arranged in concentric circle
inside a Large Tissue Tek cryomold, snap frozen in a petri dish FLOATING on
liquid nitrogen, put something underneath this dish to keep it from tipping
overor block cracks!  Cut at 5 um at -20C, pick up on Plus charge slides,
air dry for no less than 30 minutes, up to 4 hours, even overnight over 16
mesh silica gel.  Fix in reagent grade acetone 4C (refrig temp) for 10 min
- single fixation, air dry for 30 minutes, then stain. 

Another trick that really works, do everything the same way, cut frozens,
then fix in 4C acetone 10 min, air dry for 15 min, go back to 4C acetone
for 10 min, air dry for minimum of 30 min. This double fixation WILL
improve morphology but if you try to do a hydrogen peroxide endogenous
peroxidase block that has a high concentration of H202, it can chew
sections off the slide.  I use Dako's peroxidase blocker designed for
frozen sections, but better yet, use a glucose oxidase block for all
endogenous peroxidase/pseudoperoxidase block for total, complete removal on
FROZEN sections. 

Another fixation that works for most antibodies, improves morphology,
sections stay on, cut sections, air dry overnight, fix in 75% acetone/25%
absolute ethanol at RT for 5 min, rinse immediately with 3 changes of PBS.
Do glucose oxidase blocking with this fixation.  This was passed onto me by
Barb Wright.  

Perfectly flat frozen sections are a must, without curling, fat and
connective tissue on intestine creates problems and should be removed
gently to not tear holes in gut.  Be careful how you do the tissue
initially to make sure you get the best sections. 

IHC staining rinses are always done so buffer flows above where section in
mounted, never direct stream of PBS at section and cut tip of buffer bottle
so it is wider opening to give a gentle stream rather than firehose effect. 

I never lose gut sections with these methods. 


At 02:14 PM 10/5/01 -0600, you wrote:
>Help! I am futiley attempting to do an immuno on mouse intestine
>cryosections - but the sections keep leaping off the slides after I fix in
>acetone! I've tried silane, Plus slides, old-fashioned subbed
>slides....They stick after formaldehyde fixation, but the antibody we are
>using requires serious retrieval following formaldehyde (I'm doing that
>immuno right now). Anyway - at this point it is just making me crazy and
>I'd like to know how anyone gets around this nightmare. Or is it just us?
>Someone else in my department has tried this before and gave up on acetone
>- but there must be a way to get this to work!
>So - bottom line - what is the magic to getting mouse intestine
>cryosections to stick following acetone fixation?
>Many thanks,
>(a very frustrated) Tamara
> Tamara Howard
> Department of Cell Biology and Physiology
> University of New Mexico - Health Sciences Center
> Albuquerque, NM 87131
Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367
406 994-4303 (FAX)

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