[Histonet] folds forming in bone/cartilage sections

[Gehan, Loralee]

We have been using a warming plate to lay our sections on after we cut them.
This has helped them flatten out and stick to the plus slides better. 
Loralee Gehan

> ----------
> From: 	Gayle Callis
> Sent: 	Wednesday, January 14, 2004 3:05 PM
> To: 	Mike Sohaskey; Histonet <@t> lists.utsouthwestern.edu
> Subject: 	Re: [Histonet] folds forming in bone/cartilage sections
> 
> 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
> sections!   
> 
> 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:
> >Hello,
> >
> >	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.
> >
> >MIKE
> >
> >Michael Sohaskey, Ph.D.
> >University of California, Berkeley
> >Molecular and Cell Biology
> >585 Life Sciences Addition
> >Berkeley, CA  94720-3200
> >sohaskey <@t> socrates.berkeley.edu
> >(510) 643-2775 (phone)
> >
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> >
> >
> >
> Gayle Callis
> MT,HT,HTL(ASCP)
> 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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