Daniel:Protocols/RNAScope: Difference between revisions
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===Tissue Prep (~30 minutes)=== | ===Tissue Prep (~30 minutes)=== | ||
#Chill 4 %PFA (Fresh 4% PFA or 10% NBF in 1X PBS) to 4C (ice) | #Fixation | ||
#Remove slides from -80C and immediately immerse in pre-chilled fixative for '''15 minutes''' | ##Chill 4 %PFA (Fresh 4% PFA or 10% NBF in 1X PBS) to 4C (ice) | ||
#Incubate slides in 50% EtOH for 5 minutes at RT | ##Remove slides from -80C and immediately immerse in pre-chilled fixative for '''15 minutes''' | ||
#Incubate slides in 70% EtOH for 5 minutes at RT | #Dehydration | ||
#Incubate slides in 100% EtOH for 5 minutes at RT | ##Incubate slides in 50% EtOH for 5 minutes at RT | ||
#Incubate slides in fresh 100% EtOH for 5 minutes at RT | ##Incubate slides in 70% EtOH for 5 minutes at RT | ||
#Air dry on a paper towel for 5 minutes at RT | ##Incubate slides in 100% EtOH for 5 minutes at RT | ||
#Draw a hydrophobic barrier 2-4 times around each section with barrier pen. | ##Incubate slides in fresh 100% EtOH for 5 minutes at RT | ||
##Air dry on a paper towel for 5 minutes at RT | |||
#Hydrophobic Barrier | |||
##Draw a hydrophobic barrier 2-4 times around each section with barrier pen. | |||
##Let the barrier dry completely (~1 minute) | ##Let the barrier dry completely (~1 minute) | ||
#Turn on oven at set temperature to 40C; wet humidifying paper | #Turn on oven at set temperature to 40C; wet humidifying paper | ||
#Place slides on slide rack, add Pretreat 4 to entirely cover each section | #Pretreatment | ||
#Incubate for 30 minutes at RT | ##Place slides on slide rack, add Pretreat 4 to entirely cover each section | ||
##This time may be considerably shorter for different tissues, recommend 5 minutes for BA8 | ##Incubate for 30 minutes at RT | ||
##Pretreat 3 is 1/2X Pretreat 4, can provide even gentler pretreat | ###This time may be considerably shorter for different tissues, recommend 5 minutes for BA8 | ||
# Take each slide from rack and tap or flick to remove excess liquid; immediately submerge in 1X PBS | ###Pretreat 3 is 1/2X Pretreat 4, can provide even gentler pretreat | ||
#Wash slides in 1X PBS by moving rack up and down 3-5 times; repeat with fresh 1X PBS | ##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=== | ===RNA Scope Assay=== | ||
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##Air dry slides for at least 5 minutes | ##Air dry slides for at least 5 minutes | ||
#Evaluation | #Evaluation | ||
##Evaluate under standard brightfield microscope at 20-40X magnification | |||
##Positive control signal should be visible as punctate dots within cell nucleii at 20-40X magnification | |||
##Negative control may have signal; One dot to every 10 cells is acceptable | |||
##Scoring: | |||
[[Image:RNAScope_Scoring_Card.png|400px]] |
Revision as of 16:39, 25 March 2015
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)
- Fixation
- 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
- Dehydration
- 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
- Hydrophobic Barrier
- 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
- Pretreatment
- 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
- Probe Hybridization
- 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
- AMP1
- 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
- AMP2
- 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
- AMP3
- 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
- AMP4
- 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
- AMP5
- Tap or flick to remove excess liquid from slides; add ~4 drops of AMP5 to cover each section
- Incubate 30 minutes at RT
- 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
- AMP6
- Tap or flick to remove excess liquid from slides; add ~4 drops of AMP3 to cover each section
- Incubate 15 minutes at RT
- 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
- Signal Detection
- Mix equal volumes of Brown-A and Brown-B (DAB substrate) (e.g. 2 drops each); make ~120 uL DAB per section, mix well 3-5 times
- Tap or flick to remove excess liquid from slides; place ~120 uL DAB onto each section
- Incubate 10 minutes at RT
- Submerge slides in DDI water; agitate 3-5 times; replace with fresh DDI water
- Counterstain
- Submerge slides in 50% hematoxylin I solution for 2 minutes at RT; slides will be purple
- Immediately transfer slides back to DDI water; agitate 3-5 times; repeat with fresh DDI water until slides are clear and sections are purple
- Replace DDI water with 0.02% ammonia water; agitate 2-3 times; sections should turn blue
- Submerge slides in DDI water, wash 3-5 times
- Dehydrate
- Submerge slides in 70% EtOH in fume hood for 2 minutes with occasional agitation
- Submerge slides in 100% EtOH in fume hood for 2 minutes with occasional agitation
- Submerge slides in fresh 100% EtOH in fume hood for 2 minutes with occasional agitation
- Submerge slides in XYLENE for 5 minutes in fume hood with occasional agitation
- Mounting
- Remove slides from xylene and lay flat with sections up in the fume hood
- Mount slides by adding 1-2 drops of cytoseal mounting medium and seal with a coverslip; avoid air bubbles
- Air dry slides for at least 5 minutes
- Evaluation
- Evaluate under standard brightfield microscope at 20-40X magnification
- Positive control signal should be visible as punctate dots within cell nucleii at 20-40X magnification
- Negative control may have signal; One dot to every 10 cells is acceptable
- Scoring: