RNAscope assay

RNAscope assay 

Mouse brain was freshly perfused and fixed in 4% PFA for 24 hours at 4°C. One day later, the brain was immersed in 30% sucrose, allowing the tissue to sink to the bottom of the container. The tissue was frozen in the optimal cutting temperature embedding media with dry ice. The blocks were sectioned by cutting 15-μm sections. Sall1 RNA probe is commercially available from Advanced Cell Diagnostics (ACD). Here, we used probes against mouse Sall1 (ACD catalog no. 469661-C3), positive control probe (ACD catalog no. 310771) and negative probe (ACD catalog no. 310043), and then performed the assay by using the RNAscope Flurorescent Multiplex Reagent Kit (ACD catalog no. 320850) according to the manufacturer's instructions. Briefly, the fixed frozen tissue slides were post-fix by immersing them prechilled 4 % PFA in 1X PBS for 15 mins at 4°C. Following dehydrating the tissues in 50% EtOH, 70% EtOH and 100% EtOH each for 5 min at RT. Prepare fresh 1X Target Retrieval reagent (ACD catalog no. 322000) in a beaker, and maintain uniform boiling at 99-100 °C, keep the slides in the solution for 3 min. Add 4 drops of Protease III (ACD catalog no. 322340) to each section, incubating for 30 min at 40 °C, then start to run the hybridization assay:

1. Add ~4 drops of the appropriate Sall1 probe (ACD catalog no. 469661-C3) to entirely cover each section.

2. Place the slides in the humidity control tray. Close tray and insert back into the oven for 2 hrs at 40°C.

3. Wash slides in 1X Wash Buffer (ACD catalog no. 310091) twice for 2 min, each time at RT. 

4. Add ~4 drops of Amp 1-FL to entirely cover each section.

5. Place the slides in the humidity control tray. Close tray and insert back into the oven for 30 min at 40°C. 

6. Wash slides in 1X Wash Buffer twice for 2 min, each time at RT.  

7. Add ~4 drops of Amp 2-FL to entirely cover each section. 

8. Place the slides in the humidity control tray. Close tray and insert into the oven for 15 min at 40°C. 

9. Wash slides in 1X Wash Buffer twice for 2 min, each time at RT. 

10. Add ~4 drops of Amp 3-FL to entirely cover each section. 

11. Place the slides in the humidity control tray. Close tray and insert into the oven for 30 min at 40°C. 

12. Wash slides in 1X Wash Buffer twice for 2 min, each time at RT. 

13. Add ~4 drops of Amp 4-FL–Alt A to entirely cover each section. 

14. Place the slides in the humidity control tray. Close tray and insert into the oven for 15 min at 40°C. 

15. Wash slides in 1X Wash Buffer twice for 2 min, each time at RT. 

16. add ~4 drops of DAPI to each section, incubate for 30 S at RT. 

17. Remove DAPI from slides and immediately place 1–2 drops of the ProLong Gold Antifade Mountant (Invitrogen catalog no. P36930) onto each section. 

18. Carefully place a 24 mm x 50 mm coverslip over the tissue section. 

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