TITLE: BaseScope In Situ Hybridization
AUTHORS: madalynn.erb Erb

[DESCRIPTION]
BaseScope in situ hybridization on mouse brain sections

[STEPS]
SECTION: Day 1
1. Use 35μM floating brain sections
SECTION: Day 1
2. Wash sections 3 times (5 minutes) in PBS to remove cryoprotectant solution
SECTION: Day 1
3. Mount tissue onto Superfrost plus slides
SECTION: Day 1
4. Dry slides at room temp and then dip in mQ H2O to remove salt
SECTION: Day 1
5. Dry slides at 60°C for 2 hours
SECTION: Day 1
6. Let tissue dry at room temp overnight – make sure tissue is completely stuck to slides 
SECTION: Day 2
7. Dry slides at 60°C for 30 minutes
SECTION: Day 2
8. Dehydrate slides in 50%, 70%, 100% EtOH, for 5 min each at room temp
 

SECTION: Day 2
8.1. Let slides air dry for 5 min at room temp. 
SECTION: Day 2
9. Make 200mL Target Retrieval Reagent (1:10 180mL H2O + 20mL 10X Target Retrieval Reagent) 
SECTION: Day 2
10. On the bench – add 5-8 drops of Hydrogen Peroxide to tissue sections 
SECTION: Day 2
10.1. Incubate at room temp for 10 min
SECTION: Day 2
11. Rinse slides 2 times in H2O
SECTION: Day 2
12. In the steamer heat one dish of H2O and one dish of Target Retrieval Reagent
SECTION: Day 2
12.1. Check temp of Target retrieval reagent (must be at least 95°C)
SECTION: Day 2
12.2. After preheating – place slides into H2O for 10 sec to acclimate.
SECTION: Day 2
12.3. Move slides to Target Retrieval Reagent and steam for 5 min
SECTION: Day 2
13. Remove the water container and the container with slides from the steamer and place on the bench.
SECTION: Day 2
14. Wash slides in H2O for 15 sec at room temp
SECTION: Day 2
15. Wash slides in 100% EtOH for 3 min at room temp
SECTION: Day 2
16. Dry slides at 60°C for ≥5 min
SECTION: Day 2
11.1. Submerge in dish of H2O and move slides up and down 3-5 times for each wash
SECTION: Day 2
13.1. Let slides cool on the bench for 20 -30 minutes (wait until temperature of water container has dropped to 30°C)
17. Use an Immedge hydrophobic barrier pen to draw a barrier around your tissue section(s)
17.1. Let the barrier dry ≥ 1 min or overnight at room temp (you can take a break at this point)
18. Turn on RNA Scope oven – set to 40°C
18.1. Wet humidifying paper (filter paper) completely with H2O and place in the Humidity Control Tray
18.2. Warm the covered tray in the oven for 30 minutes before use
19. Place slides in slide rack and add ~5 drops of Protease IV
19.1. Incubate in pre-warmed RNAscope oven in the humidity tray at 40°C for 30 min 
20. Wash slides in H2O at room temp
21. Prepare 1L of wash buffer (20mL of 50X Wash buffer + 980mL H2O)
21.1. Heat 50X wash buffer to 40°C for 10-20 min before making 1X wash buffer
22. Equilibrate each BaseScope reagent and probe to room temp for 30 min before use

  
Important: Do not let sections dry out between incubation steps
  
All incubation steps are in the slide rack – remove slides from slide rack to wash between incubations
  
For the incubations in the HybEZ oven – cover the slide rack with the lid and make sure to lock the oven so that the water in the humidity chamber does not evaporate.
23.       Add ~4 drops of BaseScope probe to completely cover tissue
23.1. Incubate in the oven at 40°C for 2 hours
23.2. Wash 2 times in Wash Buffer at room temp - 2 min each wash
24. Add ~4 drops of AMP1 to completely cover tissue
24.1. Incubate in the oven at 40°C for 30 min
24.2. Wash 2 times in Wash Buffer at room temp - 2 min each wash
25. Add ~4 drops of AMP2 to completely cover tissue
25.1. Incubate in the oven at 40°C for 30 min
25.2. Wash 2 times in Wash Buffer at room temp - 2 min each wash
26. Add ~4 drops of AMP3 to completely cover tissue
26.1. Incubate in the oven at 40°C for 15 min
26.2. Wash 2 times in Wash Buffer at room temp - 2 min each wash
27. Add ~4 drops of AMP4 to completely cover tissue
27.1. Incubate in the oven at 40°C for 30 min
27.2. Wash 2 times in Wash Buffer at room temp - 2 min each wash
28. Add ~4 drops of AMP5 to completely cover tissue
28.1. Incubate in the oven at 40°C for 30 min
28.2. Wash 2 times in Wash Buffer at room temp - 2 min each wash
29. Add ~4 drops of AMP6 to completely cover tissue
29.1. Incubate in the oven at 40°C for 15 min
29.2. Wash 2 times in Wash Buffer at room temp - 2 min each wash
30. Add ~4 drops of AMP7 to completely cover tissue
30.1. Incubate at room temp for 15 min
30.2. Wash 2 times in Wash Buffer at room temp - 2 min each wash
30.3. Staining intensity can be modified by adjusting the AMP7 incubation time
31. Add ~4 drops of AMP8 to completely cover tissue
31.1. Incubate on the bench at room temp for 15 min
31.2. Wash 2 times in Wash Buffer at room temp - 2 min each wash
32. Prepare BaseScope Fast RED working solution (1:60 ratio)
32.1. Spin down BaseScope Fast RED-B before using
32.2. 2uL RED-B + 120uL RED-A : mix well
32.3. Use the Fast Red solution within 5 minutes – do not expose to direct sunlight or UV light
33. Add ~120µL Fast Red solution to each slide / section
33.1. Cover sections on tray to protect from light
33.2. Incubate at room temp for 10 min
33.3. Rinse 2 times in H2O at room temp
34. Dry slides at 60°C for ≥ 15 min (until slides are completely dry)
34.1. The red substrate is alcohol sensitive. DO NOT dehydrate the slides in alcohol, and make sure your reagents are not contaminated with alcohol. 
35. Place 1-2 drops of VectaMount or Ecomount on the slide and coverslip the tissue sections. 
36. Dry slides at room temp for ≥ 5 min   
29.3. We are done with the oven – but keep using the slide rack for incubations at room temp