RE: [Histonet] Processing tissue engineering sample

From:"Charles Scouten"

For more convenient chilled isopentane, see the following link:


Charles W.  Scouten, Ph.D.
5918 Evergreen Blvd. 
St. Louis, MO 63134 
Ph: 314 522 0300  
FAX  314 522 0377 

 -----Original Message-----
From: []  On Behalf Of
Sent:	Monday, August 16, 2004 11:50 AM
To:	Kobler, James
Cc:	'';
Subject:	Re: [Histonet] Processing tissue engineering sample

I have cut gels for IHC and maybe able to give you some information.
Freezing in isopentane works for us.  We do not use cryoprotection and have
cut the gels to orient them.

I would be glad to talk with you and send you some references if you would

Carol M. Johnston HT(ASCP)
M.D. Anderson Cancer Center
1515 Holcombe Blvd. Unit 443
Houston, Texas 77030

                                      "Kobler, James"                                                                        
                                             Sent by:       "''"                            
                                  08/16/2004 11:11 AM                                                                        
                                                            [Histonet] Processing tissue engineering sample                  

I would like advice about processing some experimental gels containing
fibroblasts.  The goal is to stain immunohistochemically for extracellular
collagen in small gel samples (5mm diameter, 300 micron thick).  It is
to cut the gels perpendicular to the surface in order to get multiple
that can be stained with different antibodies.  Cryosectioning would be
preferable since dehydration would probably cause shrinkage of the gels.

Questions:  (1) should the gels be treated with a cryoperservative such as
sucrose before freeezing? (2) any tips for orienting the gel discs for nice
cross-sections? (3) Is freezing in isopentane cooled with liquid nitrogen
best freezing method?

Thanks very much,

Jim Kobler, Ph.D.
Mass General Hospital

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