Tech Note: RNAscope on free floating/thick sections

If you want to use RNAScope with thicker sections than recommended (for fixed frozen tissues 7-15 um), it is possible, with adaptations in order to make sure tissue will stick well to the slide. Following these steps, you will be able to mount the sections on slides, ensuring they adhere well.

Part 1: Samples Preparation 1. Cut 30–40 µm sections. 2. Collect sections into large petri or TC dishes in 0.5x TBS +0.1% Tween20 (TBST). 3. Store sections at 4°C for no more than 2 days. (IF your samples are older than this, the RNA quality may be too low). 4. Wash free floating section with TBST once. 5. Perform the H2O2 step floating (to avoid detachment) (for Chromogenic and V2 assay) 6. After H2O2, rinse your samples with PBST and place your sections on SuperFrost Plus slides (no other brands) 7. dry at room temperature for 1 hr 8. Dip the slides in water for 3 times (<3 seconds) 9. Dry at room temperature for another 1 hr 10. Bake at 60 C for 60 min 11. Rehydrate to water 12. 100% ethanol dip 13. Dry sections (steps 7 to 13 help optimal adhesion of the sections to the slides) 14. Proceed to target retrieval (15 min, in a steamer or on a hotplate monitoring the temperature constantly) 15. After Target Retrieval, rinse your slides with mQ water, then 100% EtOH and bake sections again for 30 minutes at 60°C (also this step helps keeping sections attached to the slides) 16. Proceed with creating a barrier, then protease Plus (Chromogenic) or III (V2 assay) treatment (test 30 min, then eventually adjust if needed) and then move forward to detection steps . After protease treatment, follow with Probe incubation and detection steps, as per protocol. 17. Use the EZ-Batch System for all washes and increase washing steps to 3x5 minutes

NOTE: baking steps are done in dry oven or in HybEZ (no lid nor dehumidifying paper)

(steps 7 to 13 help optimal adhesion of the sections to the slides; H2O2 step is involved in all assays)

Part 2: RNAscope procedure

After sample preparation, you go straight to the standard protocol for Multiplex V2 for fixed frozen tissue samples. Due to the thickness of the tissue, there is risk of detachment during the pre-treatment and washing steps. For this reason, we recommend adding baking step (30min 60C in dry oven) after target retrieval. For the rest of assay you can proceed as per standard protocol.