[Histonet] Sucrose infiltration and Bone question...

[gayle callis]

Jamie, 

One thing to try is put your mouse bones under vacuum when you cryoprotect
them.   Vacuum will speed up the exchange and when you don't see bubbles
coming off bone anymore it should be infiltrated - hopefully. 

I have always wondered about using 4C temps and just did it without asking
questions.  However, I have seen replies on Histonet where RT was used. I
wonder if the rationale for 4C is that the sucrose could get some type of
unwanted growing contaminant over a longer time period. If you are not doing
IHC with chromogens, you could put sodium azide preservative in the sucrose
to counteract the growth issues.  Try suspending the bone in the sucrose so
the solution surrounds the bones totally. If you have a vacuum oven at
ambient RT it will even nicer so you don't have to deal with greasing a
vacuum dessicator.  We use O Ring Wheaton vacuum dessicators that do not
need to be greased and easy to clean. 

I suggest you access this publication and as a member of NSH, it will be
free to you if you do not have the journal on hand.  John Tarpley,
Preparation and sectioning of undecalcified frozen rodent long bones and
joints using a tape transfer system.  J Histotechnology 26(4):41, 2003.
John worked with rat bones but his method was very successful.  His
preparation protocol was quite detailed with some interesting special ways
of working with bones. If worked for him on rat bones, it may help you with
the tiny difficult mouse bones. 
Try contacting John Baker to see if he can help you too.  Info is found
below. I think he dealt with mouse bone and the Cryojane. 

bakerj <@t> umich.edu (john baker) 
John A. Baker
The University of Michigan
Orthopaedic Research Laboratories
Histology Unit
400 North Ingalls, G161
Ann Arbor, MI 48109-0486
my office 734-936-1635
lab office 734-763-9674

Good luck

Gayle Callis
HTL(ASCP)HT,MT
Bozeman MT



-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Jamie E
Erickson
Sent: Wednesday, May 20, 2009 7:32 AM
To: histonet histonet
Subject: [Histonet] Sucrose infiltration and Bone question...

Hi All,
             I have two question about sucrose infiltration....First,  I 
have infiltrated many mouse brain with sucrose 30% in the past and it has 
always been at 4degrees C (dc) and I'm asking myself now  why? My mouse 
brain are fixed in paraformaldehyde or formaldehyde for 24 hours prior so 
why sucrose @ 4dc? Would it not infiltrate better (faster)  at room temp? 
I could not find out why 4dc...

My real problem is that  I am tiring to get good mouse tibia/femur  frozen 
sections and I am using D-profile blade and a tape transfer method. It 
worked great on my rats sections but not mouse so I think the sucrose is 
not getting in to the bone.   I can't use the sinking tissue as an 
indicator because the bone sinks right away.

Should I go longer in sucrose (maybe 48 Hrs)  but at 4dc or room temp  ?

Would adding 2 % DSMO to sucrose facilitate penetration..??

Any suggestions would be great..

Thanks have a great day..

Jamie
_______________________________
Jamie Erickson
Sr. Research Associate II M.S. HTL (ASCP)
Department: DSMP
Abbott Bioresearch Center
100 Research Drive
Worcester, MA 01605-4341
508-688-3134
FAX: 508-793-4895
e-mail: jamie.erickson <@t> abbott.com




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