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