We extract the full eye from a mouse, and then fix it in 4% paraformaldehyde immediately after extraction. After punching a hole in the cornea, we fix it with 4% PFA in 20 degrees for 4 hours, and then we put the eye into a sucrose gradient 10% 20% and then 30% sucrose overnight. The tissues are put into OCT medium cryomold and sectioned onto superfrost slides. Will these tissues be acceptable for In Situ Hybridization? Do I need to refix before pretreating the tissues?
I agree with Lale Evsen.
You don't need to refix it.
Your protocol is ok to proceed to ISH.
You just have to use slice like superfrost plus or gold (or similar ) to avoid tissue detachment.
Good luck.
I use a well standardized method for in situ with formalin fixed tissue. There are a lot of protocols in literature. Of course you need to unmask the dna from linked protein and forming bridges with Proteinase k ( from loweest to highest concentrations, depending on different factors), but is works!No doubts! But this is the reason for using superfrost slices, as Virginie sayd.
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