Daniel:Protocols/RNAScope
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RNA Scope
RNA Scope is a proprietary protocol used by ACD Bio. It can be done using fluorescence (3 channels, maybe 4, +DAPI) or using chromogenic staining (2 colors plus counter stains).
Manuals
- Fresh Frozen Sample Prep User Manual
- Multiplex Fluorescent Assay User Manual
- BROWN Detection Kit Manual
- Fresh Frozen Tissue Prep Quick Guide
Chromogenic Staining Protocol
Tissue Prep (~30 minutes)
- Chill 4 %PFA (Fresh 4% PFA or 10% NBF in 1X PBS) to 4C (ice)
- Remove slides from -80C and immediately immerse in pre-chilled fixative for 15 minutes
- Incubate slides in 50% EtOH for 5 minutes at RT
- Incubate slides in 70% EtOH for 5 minutes at RT
- Incubate slides in 100% EtOH for 5 minutes at RT
- Incubate slides in fresh 100% EtOH for 5 minutes at RT
- Air dry on a paper towel for 5 minutes at RT
- Draw a hydrophobic barrier 2-4 times around each section with barrier pen.
- Let the barrier dry completely (~1 minute)
- Turn on oven at set temperature to 40C; wet humidifying paper
- Place slides on slide rack, add Pretreat 4 to entirely cover each section
- Incubate for 30 minutes at RT
- This time may be considerably shorter for different tissues, recommend 5 minutes for BA8
- Pretreat 3 is 1/2X Pretreat 4, can provide even gentler pretreat
- Take each slide from rack and tap or flick to remove excess liquid; immediately submerge in 1X PBS
- Wash slides in 1X PBS by moving rack up and down 3-5 times; repeat with fresh 1X PBS
RNA Scope Assay
- Place AMP 1-6 reagents at RT; Warm oven to 40C; warm probes for 10 minutes at 40C, swirl gently to mix
- Tap or flick to remove excess liquid from slides; place in slide rack; add ~4 drops of appropriate probe, cover the section
- Incubate for (at least) 2 hours at 40C
- Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down
- Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down
- Tap or flick to remove excess liquid from slides; add ~4 drops of AMP1 to cover each section
- Incubate 30 minutes at 40C
- Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down
- Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down
- Tap or flick to remove excess liquid from slides; add ~4 drops of AMP2 to cover each section
- Incubate 15 minutes at 40C
- Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down
- Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down
- Tap or flick to remove excess liquid from slides; add ~4 drops of AMP3 to cover each section
- Incubate 30 minutes at 40C
- Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down
- Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down
- Tap or flick to remove excess liquid from slides; add ~4 drops of AMP4 to cover each section
- Incubate 15 minutes at 40C
- Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down
- Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down