Hi Adnan,

you could use a general rehydration protocol like it is used for IHC.

You have to put your tissue in decreasing EtOH concentrations strating with two times 100% to 90%, 80%, 2 times 70% and 50% (v/v in H2O).

Afterwards you should be able to transfer the tissue in any aqueous buffer that is needed.

For already cutted slides you would normally use 5min per incubation step. Depending on the thickness of your tissue you should increase the time.

Best

Lukas

Dear Lukas, 

You meant to say methanol ? do you any standard protocol ?

Hi Adnan,

you're right, in your case it would be methanol with lowering concentrations.

There is no special protocol for that, it's a general technique that is also used for paraffin-embedded tissues.

Hi Adnan, I recommand do to paraffin embedding because of the long duration in 100% methanol. Put your piece of tissue in Isopropanol 2x 60 min, Xylene 2 x 45 min, Xylene/Paraffin (à/à) 45 min at 60°C, Paraffin 2 x 3 h  at 60°C or the second Paraffin bath over night. After blocking the tissue you can cut it between 3 - 10 µm. For your ISH protocol  you might check weather a protease pretreatment is necessary.

I second Ute's recommendation. Cutting will be a pain of tissue, that is dehydrated this long time. And usually rehydration does not bring it back to native status.

Thank you very much Ute, i will try this method out 

I do not really agree with Ute and Gudrun.

Why embedding the tissue in parafin when you have to rehydrate them afterwards again for the ISH? For normal tissue that should be used for IHC to detect proteins this would be a suitable way for longer storage. But even then I would embed them as fast as possible instead of keeping them in the fixiative.

In the case of (F)ISH it really depends on what you are looking at (RNA or DNA)?Generally I would freeze down the tissue as soon as possible and keep it in OCT at -80 instead of storing them in the fixiative.

It also depends on the kit/assay you're using. Some of them does not work in paraffin embedded tissue at all.