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.scou...@leica-microsystems.com
[mailto:charles.scou...@leica-microsystems.com]
Sent: Wednesday, February 24, 2010 2:03 PM
To: ndev...@stanford.edu; histonet@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/
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From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Nicholas
David Evans ndev...@stanford.edu
Sent: Wednesday, February 24, 2010 12:34 PM
To: histonet histonet@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
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