Day 1:

1) Air dry slides for at least 30 minutes

2) Fix tissue in fresh 4% PFA in PBS for 10 to 15 minutes

3) ProtK in PBS (1 ug/ml: 1 ul into 20 ml) for 5 to 10 minutes depending on thickness of sections (for 14 um thick, I do 5 minutes)

4) Rinse in PBS 2x

5) Refix in PFA for 5 minutes at RT

6) Ethanol dehydration 5 minutes each: 50%, 70%, 100%, 100% Ethanol

7) Let slides air dry for 5 minutes at RT

8) Put slides in probe hybridization buffer at 37C for at least 10 minutes

9) Prepare probe solution (0.4 ul of 2 uM stock HCR probe into 100 ul of pre-warmed probe buffer). Cover sections in parafilm.

10) Incubate slides at 37C overnight in a moist chamber

Day 2:

1) Remove excess probe by washing 4 x 5 minutes with wash buffer at 37C

2) Wash samples 2 x 5 minutes with 5X SSCT (0.1% Tween) at room temperature

3) Put samples in amplification buffer for 30 minutes at room temperature

4) Prepare hairpins separately - 2 ul of 3 uM stock per 100 ul volume of amplification buffer

- heat at 95C for 90 seconds and cool to room temperature in a dark drawer for 30 minutes

5) Add snap cooled hairpins to 100 ul of room temperature amplification buffer

6) Remove amplification buffer and add hairpins. Cover slides in parafilm. Leave slides incubating in the dark in a humidified chamber overnight.

Day 3:

1) Wash slides in 3 x 5 min in 5x SSCT at room temperature

2) Wash slides in 5x SSC at room temperature

3) Remove 5x SSC, and airdry the slide for no more than 5 minutes. Add antifade mounting reagent and cover slip.