Re: [Histonet] folds forming in bone/cartilage sections

From:Gayle Callis

Two things that may help

One, be careful about overprocessing mouse bone, decalcified tibia need
only 1 holur per change, with this schedule and hopefully you have vacuum
and pressure.  

70%, 80%, 95% X 2, 100% x 2, xylene X 1, Clearite 3 X 1, paraffin a minimum
of 3 chnages (30, 30, 1 hour)  Use harder paraffin to give more rigidity to
support embedding media, Tissue Prep 2 is excellent or at least embed in
this. Do not add heat to processing and make sure the paraffin is no hotter
than 60C.  If you remove too much of bound water from cartilage rather than
free water in tissue spaces, you will have problems.  What you are dealing
with is softer growth plate cartilage surrounded by much harder bone.
Soaking helps the harder decalcified bone soften while cartilage is picking
up some water. Don't oversoak or bone will swell out of block = messy

Trim block, soak on ice block with water on top of ice for a few minutes,
cut at 5 um using a really GOOD quality disposable blade, and lay ribbon on
a waterbath containing approx 5% DMSO! Pick up section on plus charge
slide, drain well and dry overnight at 37C.  If your waterbath is too hot,
wrinkles set and you cannot get them corrected. Adjust waterbath carefully.  

DMSO lowers the surface tension of water, and allows cartilage to stretch
properly without having to pull on the paraffin ribbon.  Your ribbon must
be totally uncompressed and wrinkled to begin with and that requires the
best blade.  I have excellent luck with these knives, Dura Edge, AccueEdge
and EdgeRite.  We also prefer high profile for more stability, edges are as
sharp as low profiles. 5 um sections are preferred, 10 um may give you more
wrinkle problems.  Low profile blades will work as long as the bones are
perfectly processed. 

Good luck,

At 10:36 AM 1/14/2004 -0800, you wrote:
>	I find that, when sectioning EDTA-decalcified mouse tibiae at 
>5 - 10 microns, that folds (i.e., creases) frequently form 
>specifically within the growth plate cartilage as the sections dry 
>and adhere to the Superfrost slides.  I have had no success with 
>increasing the amount of time that I float the sections on a water 
>bath prior to drying them on slides.  I'm hoping that someone might 
>be able to offer a suggestion as to how I can overcome this 
>difficulty.  Thanks very much.
>Michael Sohaskey, Ph.D.
>University of California, Berkeley
>Molecular and Cell Biology
>585 Life Sciences Addition
>Berkeley, CA  94720-3200
>(510) 643-2775 (phone)
>Histonet mailing list
Gayle Callis
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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