[Histonet] Coverslipping Problem - Please Help!

Brian Rabe rabe_brian <@t> yahoo.com
Thu Jul 29 17:14:12 CDT 2010

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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