Hallo Ruth, generally I would recommand to establish the method on tissue that expresses your gene you are looking for. It makes the evalution of your results easier. Keyword pre-Hybridisation. If you are working with Kryosections there is no need to fix the tissue bevor cutting. Prepare native cryosections air dry them for 30 min and put them in to the -80°C-fridge until you do the ISH. After equilibration to room temperature (1 hour) start with the fixtion in ice cold 4% PFA for 10 min. (may be shorter; it depends on the thicknes of your sections; this is a protocol for 20 µm thick brain sections); rinse in 2xSSC for 5 min; Acetic Anhydride for 10 min; 4x1 min 2xSSC; 5min 2XSSC; 30 min Prehybridisation Buffer (we use a ready-to- use buffer from Sigma-Aldrich) and then add your probe for hybridisation. The temperature and the duration of your hybridisation depends on the CG-contents of your probe. You can calculate it or if you have found references in the literature, try them. To reduce autoflorescence you can add a Sudan Black stain after the detection step- Good luck!