[Histonet] Coverslipping Problem - Please Help!

[Collette, Nicole M.]

Hi, Brian,

It is likely to be a pH issue. If you use BM Purple as your detection chromogen (which is what we use in our lab for whole mounts on X. laevis and X.tropcialis, and mouse embryos for WMISH), then you must keep the embryos at acid pH from BM Purple step onwards, or you will re-activate the BM Purple (hint: Alkaline phosphatase substrate). Light from the microscope compounds your problem. You are likely cryoprotecting and/or coverslipping with neutral or slightly alkaline solution, if you use something acidic, you should be fine. You can adjust the pH of your sucrose pretty readily, so that's not too hard. If you are only coverslipping temporarily for photos (not for archiving), you can simply use a glycerol/PBS pH 5.5 mixture for photos, then discard your slides. Coverslipping for archiving might be a question better addressed my someone else on histonet who is more knowledgeable about mounting media.

Good luck!
Sincerely,
Nicole Collette
LLNL/UC Berkeley

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Brian Rabe
Sent: Thursday, July 29, 2010 3:14 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Coverslipping Problem - Please Help!

Dear Histonet members,
     I have been cryosectioning Xenopus laevis embryos this summer that have undergone a whole mount chromogenic in situ.  The embryos are then fixed overnight in a formaldehyde solution (1X MEMFA) at 4 C, then stored in 1 x PBS until they are cryoprotected in a sucrose solution at least overnight at 4 C.  Then they are put in TBS tissue freezing medium for 2-3 hours at room temperature before being frozen and sectioned.  The sections come out looking very nice with good morphology, but when they are coverslipped (2 1 minute washes in 1 x PBS to dissolve the tissue freezing medium) with VectaMount AQ aqueous mounting medium, the sections turn very dark under the microscope, especially the gut region.  They are so dark that it becomes difficult to see the signal from the in situ.  Any help or advice would be GREATLY appreciated!
Thank you for your time,
Brian Rabe
The College of William and Mary


      

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