TITLE: v2 RNAscope in situ hybridization 
AUTHORS: Andrew J. Flores

[DESCRIPTION]
This is a protocol for performing RNAscope‱ in situ hybridization analysis on fixed-frozen mouse brain tissue using the RNAscope‱ Multiplex Fluorescent v2 kit (Advanced Cell Diagnostics; ACD). This RNAscope‱ protocol similar to the v2 protocol provided by ACD; places where it deviates from ACD's protocol have been indicated. This protocol also includes steps for sample preparation (including cryosectioning) and imaging of slides using a slide scanner.  

[STEPS]
SECTION: Cryosectioning, sample preparation, and storage
1. Collect sections spaced 20 μm apart between Bregma = +1.0–0.0 in RNase-free PBS.
SECTION: Cryosectioning, sample preparation, and storage
2. Immediately mount sections onto Superfrost‱ plus microscopy slides. Mount sections spaced 120 microns apart 4 per slide (Note that this is different from the ACD protocol, which recommends mounting only 1 section per slide). This amounts to a total of 8 sections between Bregma = +1.0–0.0 mounted onto 2 slides.
SECTION: Cryosectioning, sample preparation, and storage
3. Allow slides to dry for 60 minutes at -20°C, then, immediately store at −80°C in a slide box placed inside of a Ziploc bag with drierite desiccant until the day of the RNAscope procedure.
SECTION: Pretreatment of fixed-frozen tissue samples
4. Baking. Bake slides for 60 min at 60°C in the HybEZ‱ oven. Note that this is longer than the time recommended in the ACD v2 protocol.
SECTION: Pretreatment of fixed-frozen tissue samples
5. Post-fixation. Post-fix the slides by immersing them in 4% PFA in 1X PBS for 30 min at 4°C. Note that this post-fixation time is longer than the time recommended in the ACD v2 protocol.
SECTION: Pretreatment of fixed-frozen tissue samples
6. Serial dehydration. Slides are then rinsed once in ddH2O and then serially dehydrated using an ethanol series in the following  order: 50%, 70%, two times 100% ethanol (5 min each). 
SECTION: Pretreatment of fixed-frozen tissue samples
7. Hydrogen peroxide treatment. Take the dehydrated slides and add ~5–8 drops of RNAscope Hydrogen Peroxide to cover the sections on each slide. Treat sections with hydrogen peroxide (CAT no) for 10 minutes at RT. 
SECTION: Pretreatment of fixed-frozen tissue samples
7.1. Rinse slides with ddH2O. Repeat with fresh ddH2O water.
SECTION: Pretreatment of fixed-frozen tissue samples
8. Manual target retrieval. Perform manual target retrieval using Target Retrieval Reagents () as described in Appendix B of the ACD v2 protocol.
SECTION: Pretreatment of fixed-frozen tissue samples
8.1. Prepare 700 mL of fresh RNAscope 1X Target Retrieval Reagents by adding 630 mL ddH2O to 1 bottle (70 mL) 10X Target Retrieval Reagents in a clean 1 L beaker. Mix well with stir bar on stir plate. 
SECTION: Pretreatment of fixed-frozen tissue samples
8.2. Place the beaker containing RNAscope 1X Target Retrieval Reagents on the hot plate. Cover the beaker with foil, and turn the hot plate on high until 1X Target Retrieval Reagents reaches 98–102°C.
SECTION: Pretreatment of fixed-frozen tissue samples
8.3. With a pair of forceps, slowly submerge a Tissue-Tek Slide Rack containing the slides into RNAscope 1X Target Retrieval Reagents solution. Cover the beaker with foil and boil the slides for 5 min.
SECTION: Pretreatment of fixed-frozen tissue samples
8.4. Wash slides 3–5 times by moving the Tissue-Tek Slide Rack up and down in the distilled water. Then Transfer the slides to 100% ethanol for 3 min.
SECTION: Pretreatment of fixed-frozen tissue samples
9. Slides are then air dried for 5 min and a hydrophobic barrier is drawn around sections using an ImmEdgeTM Pen (H-4000; Vector Laboratories, Inc.).
SECTION: Pretreatment of fixed-frozen tissue samples
10. Incubate slides with Protease III (ACD) reagent for 30 min at 40°C in the HybEZ‱ oven.
SECTION: Pretreatment of fixed-frozen tissue samples
10.1. Wash slides 2x with ddH2O.
14. (Optional stopping point). You can store the slides in 5X SSC overnight at RT. 
SECTION: Amplification
15. Hybridize AMP 1. Add RNAscope Multiplex FL v2 Amp1 to each slide.
SECTION: Probe Hybridization
12. Add probe mixture to slides. Refer to Appendix C. Reagent Volume Guidelines to determine the volume of probe mixture to add to each slide. 
SECTION: Probe Hybridization
13. Insert into HybEZ‱ oven for 2 hrs at 40°C.
SECTION: Probe Hybridization
11. Prepare probe mixture. C1 probes are 1X; C2 and C3 probes come as 50X solutions and therefore must be diluted 1:50 with the C1 probe.
SECTION: Amplification
16. Hybridize AMP 2. Add RNAscope Multiplex FL v2 Amp2 to each slide.
SECTION: Amplification
15.1. Incubate in the HybEZ‱ oven for 30 min at 40°C.
SECTION: Amplification
15.2. Wash slides 2x for 2 min at RT with 1X Wash Buffer.
SECTION: Amplification
16.1. Incubate in the HybEZ‱ oven for 30 min at 40°C.
SECTION: Amplification
16.2. Wash slides 2x for 2 min at RT with 1X Wash Buffer.
SECTION: Amplification
17. Hybridize AMP 3. Add RNAscope Multiplex FL v2 Amp3 to each slide Incubate in the HybEZ‱ oven for 15 min at 40°C.
SECTION: Amplification
17.1. Incubate in the HybEZ‱ oven for 15 min at 40°C.
SECTION: Amplification
17.2. Wash slides 2x for 2 min at RT with 1X Wash Buffer.
SECTION: Develop HRP Signals
18. Develop HRP-C1 signal. Add RNAscope Multiplex FL v2 HRP-C1 to slides.    
SECTION: Develop HRP Signals
18.2. Wash slides 2x for 2 min at RT with 1X Wash Buffer.
SECTION: Develop HRP Signals
18.1.  Incubate in the HybEZ‱ oven for 15 min at 40°C.
SECTION: Develop HRP Signals
19. Add 150-200 μL of fluorophore to label the C1 probe.
SECTION: Develop HRP Signals
19.1. Incubate in the HybEZ‱ oven for 30 min at 40°C.
SECTION: Develop HRP Signals
19.2. Wash slides 2x for 2 min at RT with 1X Wash Buffer.
SECTION: Develop HRP Signals
20. Add RNAscope Multiplex FL v2 HRP blocker to slides.
SECTION: Develop HRP Signals
20.1. Incubate in the HybEZ‱ oven for 15 min at 40°C.
SECTION: Develop HRP Signals
20.2. Wash slides 2x for 2 min at RT with 1X Wash Buffer.
SECTION: Develop HRP Signals
21. Develop HRP-C2 signal. Add RNAscope Multiplex FL v2 HRP-C2 to slides.    
SECTION: Develop HRP Signals
21.1.  Incubate in the HybEZ‱ oven for 15 min at 40°C.
SECTION: Develop HRP Signals
21.2. Wash slides 2x for 2 min at RT with 1X Wash Buffer.
SECTION: Develop HRP Signals
22. Add 150-200 μL of fluorophore to label the C2 probe.
SECTION: Develop HRP Signals
22.1.  Incubate in the HybEZ‱ oven for 30 min at 40°C.
SECTION: Develop HRP Signals
22.2. Wash slides 2x for 2 min at RT with 1X Wash Buffer.
SECTION: Develop HRP Signals
23. Add RNAscope Multiplex FL v2 HRP blocker to slides.
SECTION: Develop HRP Signals
23.2. Wash slides 2x for 2 min at RT with 1X Wash Buffer.
SECTION: Develop HRP Signals
23.1.  Incubate in the HybEZ‱ oven for 15 min at 40°C.
SECTION: Develop HRP Signals
24. Develop HRP-C3 signal. Add RNAscope Multiplex FL v2 HRP-C3 to slides.
SECTION: Develop HRP Signals
24.1.  Incubate in the HybEZ‱ oven for 15 min at 40°C.
SECTION: Develop HRP Signals
24.2. Wash slides 2x for 2 min at RT with 1X Wash Buffer.
SECTION: Develop HRP Signals
26. Add RNAscope Multiplex FL v2 HRP blocker to slides.
SECTION: Develop HRP Signals
26.1. Incubate in the HybEZ‱ oven for 15 min at 40°C.
SECTION: Develop HRP Signals
26.2. Wash slides 2x for 2 min at RT with 1X Wash Buffer.
SECTION: Develop HRP Signals
27. Counterstain and mount slides. Add enough DAPI to cover each section and incubate for 30 sec at RT.    
SECTION: Develop HRP Signals
27.1. Remove DAPI and place 1-2 drops of ProLong Gold Antifade Mountant on each slide
SECTION: Develop HRP Signals
27.2. Carefully place a 24 mm x 50 mm glass coverslip over the tissue section.
SECTION: Develop HRP Signals
27.3. Dry slides 30 min to overnight in the dark and store slides in the dark at 2-8°C.
SECTION: Develop HRP Signals
25. Add 150-200 μL of fluorophore to label the C3 probe.
SECTION: Develop HRP Signals
25.1. Incubate in the HybEZ‱ oven for 30 min at 40°C.
SECTION: Develop HRP Signals
25.2. Wash slides 2x for 2 min at RT with 1X Wash Buffer.