I would like to de-embed fixed tissue that are stored at -80° in OCT resin.
Final aim is to perform a whole mount immunostain with.
Does anyone have experienced such a procedure?
I would appreciate feedback or troubleshooting advices.
many thanks
OCT is water soluble. You shouldn't have any issues washing it off and subsequently staining following a normal permeabilization/blocking procedure. The whole mount staining procedure is, however, tissue dependent (wash and stain times/concentration of TritonX or other detergent etc.). More details are needed to inform on the staining side of things. Good luck!
Thanks Elliot
For the whole mount staining procedure I am pretty confident with the protocol we use in the lab.
My point was mainly for the OCT washing procedure. Do you think I should wash in PBS or should I add sucrose as I did before OCT embedding?
Thomas Jungas I would just wash with PBS. It's similar to when you cryosection--- washing the OCT off is no issue prior to staining. The only difference is the quantity. You will likely have to do multiple or extended washes to get it all off. If the tissue is already fixed, you should be good to go with the staining! Adding glucose wouldn't be necessary as you won't be freezing again. The sucrose is meant to prevent crystallization/damage of the tissue during the freezing process. Best of luck!
E
Many thanks for your time
I'll go according your recommandation.
Thomas
I did it and it worked perfectly!
I leave my frozen samples on the bench for 15 minutes and, once thawed, I wash them in PBS and perform my classical immuno-staining procedure.
The tissues were perfectly preserved.
Thanks again Elliot.
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