TITLE: Manual Basescope Duplex Assay
AUTHORS: Hector Martell Martinez

[DESCRIPTION]
This protocol details the manual basescope duplex assay.

[STEPS]
SECTION: Day 1
1. Bake slides @ 60 °C for 60 min.

SECTION: Day 2
9. Turn on oven to 40 °C. Spray everything with RNase Away.

Wash slides in 1X Wash buffer for 2 min 2X.

Wash slides in 1X Wash buffer for 2 min (1/2).
Wash slides in 1X Wash buffer for 2 min (2/2).

SECTION: Day 1
2. Deparaffinize 
SECTION: Day 1
4. Target Retrieval
SECTION: Day 1
5. Hydrophobic Barrier 
SECTION: Day 1
6. Protease III
SECTION: Day 1
7. Hybridize Probes
SECTION: Day 1
8. Wash
SECTION: Day 2
10. Cover tissue with AMP1 – incubate for 30 min @ 40 °C.

Wash slides in 1X Wash buffer for 2 min 2X.

Wash slides in 1X Wash buffer for 2 min (1/2).
Wash slides in 1X Wash buffer for 2 min (2/2).

SECTION: Day 2
11. Cover tissue with AMP2 – incubate for 30 min @ 40 °C.

Wash slides in 1X Wash buffer for 2 min 2X.

Wash slides in 1X Wash buffer for 2 min (1/2).
Wash slides in 1X Wash buffer for 2 min (2/2).
SECTION: Day 2
12. Cover tissue with AMP3 – incubate for 15 min @ 40 °C.

Wash slides in 1X Wash buffer for 2 min 2X.

Wash slides in 1X Wash buffer for 2 min (1/2).
Wash slides in 1X Wash buffer for 2 min (2/2).
SECTION: Day 2
13. Cover tissue with AMP4 – incubate for 30 min @ 40 °C.

Wash slides in 1X Wash buffer for 2 min 2X.

Wash slides in 1X Wash buffer for 2 min (1/2).
Wash slides in 1X Wash buffer for 2 min (2/2).
SECTION: Day 2
14. Cover tissue with AMP5 – incubate for 30 min @ 40 °C.

Wash slides in 1X Wash buffer for 2 min 2X.

Wash slides in 1X Wash buffer for 2 min (1/2).
Wash slides in 1X Wash buffer for 2 min (2/2).
SECTION: Day 2
15. Cover tissue with AMP6 – incubate for 15 min @ 40 °C.

Wash slides in 1X Wash buffer for 2 min 2X.

Wash slides in 1X Wash buffer for 2 min (1/2).
Wash slides in 1X Wash buffer for 2 min (2/2).
SECTION: Day 2
16. AMP 7 & 8 are @ Room temperature

Cover tissue with AMP7 – incubate for 30 min @ 40 Room temperature.

Wash slides in 1X Wash buffer for 2 min 2X.

Wash slides in 1X Wash buffer for 2 min (1/2).
Wash slides in 1X Wash buffer for 2 min (2/2).
SECTION: Day 2
17. Cover tissue with AMP8 – incubate for 15 min @ 40 Room temperature.

Wash slides in 1X Wash buffer for 2 min 2X.

Wash slides in 1X Wash buffer for 2 min (1/2).
Wash slides in 1X Wash buffer for 2 min (2/2).
SECTION: Day 2
18. Detect Red signal
SECTION: Day 2
19. AMP 9 & 10 @ 40 °C.

Cover tissue with AMP9 – incubate for 15 min @ 40 °C.

Wash slides in 1X Wash buffer for 2 min 2X.

Wash slides in 1X Wash buffer for 2 min (1/2).
Wash slides in 1X Wash buffer for 2 min (2/2).

SECTION: Day 2
20. Cover tissue with AMP10 – incubate for 15 min @ 40 °C.

Wash slides in 1X Wash buffer for 2 min 2X.

Wash slides in 1X Wash buffer for 2 min (1/2).
Wash slides in 1X Wash buffer for 2 min (2/2).

Turn off Oven!!!! Rest of protocol is at Room temperature.

SECTION: Day 2
21. Cover tissue with AMP11 – incubate for 30 min @ 40 Room temperature.

Wash slides in 1X Wash buffer for 2 min 2X.

Wash slides in 1X Wash buffer for 2 min (1/2).
Wash slides in 1X Wash buffer for 2 min (2/2).
SECTION: Day 2
22. Cover tissue with AMP12 – incubate for 15 min @ 40 Room temperature.

Wash slides in 1X Wash buffer for 2 min 2X.

Wash slides in 1X Wash buffer for 2 min (1/2).
Wash slides in 1X Wash buffer for 2 min (2/2).
SECTION: Day 2
23. Detect Green Signal
SECTION: Day 2
24. Counterstain: if hematoxylin is too dark, it will mask signal; especially green).
SECTION: Day 2
25. Dry Slides.

Dry for 15 min @ 60 °C.

SECTION: Day 2
26. Mounting.
SECTION: Day 1
1.1. After baking, wet humidifying paper, warm oven and slide tray to 40 °C.
SECTION: Day 1
3. Hydrogen Peroxide
SECTION: Day 1
2.1. Xylene – 5 min w/ agitation.

Meanwhile… turn steamer on, make 1X Target Retrieval solution, turn slide warmer to 60 °C.

SECTION: Day 1
2.2. Fresh Xylene – 5 min w/ agitation.
SECTION: Day 1
2.3. 100% EtOH – 2 min w/ agitation.
SECTION: Day 1
2.4. Fresh 100% EtOH –  2 min w/ agitation.
SECTION: Day 1
2.5. Dry on slide warmer – 60 °C for 5 min.

SECTION: Day 1
3.1. Cover tissue with hydrogen peroxide and incubate covered for 10 min.

Meanwhile… Boil 1X Target Retrieval solution in microwave then place in steamer.

SECTION: Day 1
3.2. Tap solution off on absorbent paper.
SECTION: Day 1
3.3. Wash in nanopure water (up and down 3-5 times then incubate for 2 min) 2X.

Wash in nanopure water (up and down 3-5 times then incubate for 2 min ) (1/2).
Wash in nanopure water (up and down 3-5 times then incubate for 2 min ) (2/2).

SECTION: Day 1
4.1. Transfer samples to 99 °C 1X Target Retrieval solution for 30 min.

SECTION: Day 1
4.2. Wash in nanopure water (up and down 3-5 times then incubate for ~15 s)

SECTION: Day 1
4.3. Transfer to second container of 100% EtOH in fume hood for 3 min.

SECTION: Day 1
4.4. Dry on slide warmer – 60 °C for 5 min.

SECTION: Day 1
5.1. Create barrier with hydrophobic pen – let dry for 5 min @ Room temperature.

Possible stopping point: Dry O/N @ Room temperature.

SECTION: Day 1
6.1. Cover tissue with Protease III for mouse or cell pellets. Protease IV for human tissues.
SECTION: Day 1
6.2. Incubate for 30 min @ 40 °C.

Meanwhile… Warm probes to 40 °C inside oven for ~10 min.

SECTION: Day 1
6.3. Wash in nanopure water w/ slight agitation 2X.
SECTION: Day 1
7.1. Cover tissue (~120 µL) with 1X probe mix (see below).

C1 probes are 1X; C2 probes are 50X.

Dilute C2 probe using C1 probes to 1X.

SECTION: Day 1
7.2. Incubate for 120 min @40 °C.

SECTION: Day 1
8.1. Wash in 1X Wash buffer for 2 min 2X.

Store O/N in 5X SSC @ Room temperature.

SECTION: Day 2
18.1. Make enough solution for each sample.

 2 µL Red-B + 120 µL Red-A = 1 sample (Light sensitive).

SECTION: Day 2
18.2. Cover tissue with solution – incubate for 10 min @ Room temperature.

SECTION: Day 2
18.3. Wash slides in 1X Wash buffer for 2 min 2X.

Wash slides in 1X Wash buffer for 2 min (1/2).
Wash slides in 1X Wash buffer for 2 min (2/2).
SECTION: Day 2
23.1. Make enough solution for each sample.

2.4 µL Green-B + 120 µL Green-A = 1 sample (Light sensitive).

SECTION: Day 2
23.2. Cover tissue with solution – incubate for 10 min @ Room temperature.

SECTION: Day 2
23.3. Wash slides in 1X Wash buffer for 5 min.

SECTION: Day 2
23.4. Rinse slides in nanopure water.
SECTION: Day 2
24.1. Incubate slides in 50% hematoxylin staining solution for 30 s – 1 min.

Slides should turn purple.
SECTION: Day 2
24.2. Take out immediately and wash in nanopure water 3-5X.

Until slides are clear.
SECTION: Day 2
24.3. Dip slides up and down 2-3X in 0.02% ammonia water (should turn blue).
SECTION: Day 2
24.4. Wash in nanopure water 3-5X.
SECTION: Day 2
26.1. Dip quickly in FRESH xylene
SECTION: Day 2
26.2. Place 1-2 drops of VectaMount on the slide.
SECTION: Day 2
26.3. Coverslip.
SECTION: Day 2
26.4. Air-dry.