[Histonet] M.O.M frozen section background

Thu Feb 25 11:28:16 CST 2010

Sorry, embed in OCT in a mould sitting in a slurry. Thanks for the useful
advice everyone.

nick

  _____  

From: Charles.Scouten <@t> leica-microsystems.com
[mailto:Charles.Scouten <@t> leica-microsystems.com] 
Sent: Thursday, February 25, 2010 9:26 AM
To: ndevans <@t> stanford.edu; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] M.O.M frozen section background

I would say that dehydrating and rehydrating worked by washing away the
leaked cytosol, but that you did not get as strong a stain inside the
cells as if you had cut with a vibrating microtome.  But I would agree
that sucrose cryoprotection, then immersion in a slurry with dry ice would
snap freeze as fast as it can be done, but wait, methanol?  That will
penetrate the tissue, and has its own coagulant fixative effect, no?  Did
you immersion freeze in methanol?

Cordially,

Charles W. Scouten, Ph.D

Product Manager, MNL

Biosystems Division

Leica Biosystems Richmond, Inc.
5205 Route 12
P.O. Box 528
Richmond, IL 60071
United States of America

Telephone 630 964 0501

facsimile +1 630 964 0576

www.MyNeuroLab.com <http://www.myneurolab.com/> 

www.leica-microsystems.com <http://www.leica-microsystems.com/> 

IMPORTANT - This email and any attachments may be confidential. Any
retransmissions, dissemination or other use of

these materials by persons or entities other than the intended recipient
is prohibited. If received in error, please contact

us and delete all copies. Before opening or using attachments, check them
for viruses and defects. Our liability is limited

to resupplying any affected attachments. [Any representations or opinions
expressed in this email are those of the

individual sender].

From: Nicholas David Evans <ndevans <@t> stanford.edu> [mailto:Nicholas David
Evans <ndevans <@t> stanford.edu>] 
Sent: Wednesday, February 24, 2010 7:01 PM
To: <Charles.Scouten <@t> leica-microsystems.com>;
<histonet <@t> lists.utsouthwestern.edu>
Subject: RE: [Histonet] M.O.M frozen section background

You made me worried there for a minute, Charles - I've spent a lot of time
cryosectioning rather than using PPFE! But luckily dehydrating,
rehydrating and post-fix seemed to completely get rid of the background I
was experiencing. No idea why it works, but it did. (We cryoprotect In
sucrose, then embed in methanol in dry ice).

Best wishes

Nick

  _____  

From: Charles.Scouten <@t> leica-microsystems.com
[mailto:Charles.Scouten <@t> leica-microsystems.com] 
Sent: Wednesday, February 24, 2010 2:03 PM
To: ndevans <@t> stanford.edu; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] M.O.M frozen section background

You must be using a stain for a protein or molecule present in cytosol.
The frozen sections are different that the paraffin because the freezing
process was too slow.  Ice crystals formed, and broke membranes.  Cytosol
leaked into the intracellular space, and stained there.  Background.  

Snap freeze in less than 3 seconds to solid, and the crystals cannot form.
Full Immersion in isopentane (flammable) mixed into a slurry with dry ice
would do it.

Cordially,

Charles W. Scouten, Ph.D

Product Manager, MNL

Biosystems Division

Leica Biosystems Richmond, Inc.
5205 Route 12
P.O. Box 528
Richmond, IL 60071
United States of America

Telephone 630 964 0501

facsimile +1 630 964 0576

www.MyNeuroLab.com <http://www.myneurolab.com/> 

www.leica-microsystems.com <http://www.leica-microsystems.com/> 

IMPORTANT - This email and any attachments may be confidential. Any
retransmissions, dissemination or other use of

these materials by persons or entities other than the intended recipient
is prohibited. If received in error, please contact

us and delete all copies. Before opening or using attachments, check them
for viruses and defects. Our liability is limited

to resupplying any affected attachments. [Any representations or opinions
expressed in this email are those of the

individual sender].

From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Nicholas
David Evans <ndevans <@t> stanford.edu>
Sent: Wednesday, February 24, 2010 12:34 PM
To: histonet <histonet <@t> lists.utsouthwestern.edu>
Subject: [Histonet] M.O.M frozen section background

Hi All, 

Does anyone have any tips on reducing background staining when attempting
to detect mouse monoclonal antibodies on mouse tissue? We currently use
Vector's M.O.M kit - it works great on PPFE tissue with nice specific
staining after pepsin unmasking, but in frozen tissue the background is
much higher than the signal. 

I routinely use 4% PFA pre-fix, then 3% H2O2 in MeOH for 3 mins, then all
the blocks that come with teh kit. I am using several different
cytokeratin Abs on 10 um frozen mouse sections. 

The only thing that I could think to try was to dehydrate the tissues,
rehydrate and try again with the unmasking, but if anyone has a better
idea or commonly deals with this problem, please let me know. 

Best wishes 
Nick 

_______________________________________________ 
Histonet mailing list 
Histonet <@t> lists.utsouthwestern.edu 
http://lists.utsouthwestern.edu/mailman/listinfo/histonet 

______________________________________________________________________
This email has been scanned by the MessageLabs Email Security System.
For more information please visit http://www.messagelabs.com/email 
______________________________________________________________________