More on tricks to prevent RE: [Histonet] folds forming in bone/cartilage sections

[Gayle Callis]

I have done this in the past and exploded my bone sections to the point of
no return, it also will put "bubble" artifact aka vacuole in the nuclei of
cells IF the hot plate is too warm. 

Another trick is to have a room temperature waterbath containing 10%
ethanol, lay section on that, pick up section, onto a nonplus charge slide,
then go to a warm waterbath and slowly lower section onto warmer water,
this will flatten sections.  In a pinch, I have also put a few drops of 10%
under the section after picking it up, then laying it on a warm water bath
to flatten.  Most of the time, a perfectly processed piece of bone has few
to no problems and you get the perfectly flat section with a very sharp
disposable blade.  Have been known to change blade edges between each
ribbon IF problems occur.  The price of a blade is cheap if I don't have to
constantly correct fold, compression, other problems with dicey,
frustrating bone sections. 

By the way, this has all been discussed before on Histonet, archives are
searchable for more information. 

  At 09:24 AM 1/15/2004 -0500, you wrote:
>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)
>> >
>> >_______________________________________________
>> >Histonet mailing list
>> >Histonet <@t> lists.utsouthwestern.edu
>> >http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>> >
>> >
>> >
>> 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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>> 
>
>
>
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)