Daniel:Notebook/RNAFISH/2015-3-26: Difference between revisions
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##Air dry on a paper towel 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) | #Draw a hydrophobic barrier 2-4 times around each section with barrier pen; Let the barrier dry completely (~1 minute) | ||
#Turn on oven | #Turn on oven and set temperature to 40C; wet humidifying paper | ||
#Pretreatment | #Pretreatment | ||
##Place slides on slide rack, add Pretreat 4 to entirely cover each section | ##Place slides on slide rack, add Pretreat 4 to entirely cover each section |
Revision as of 17:48, 26 March 2015
RNA Scope Chromogenic
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
- Draw a hydrophobic barrier 2-4 times around each section with barrier pen; Let the barrier dry completely (~1 minute)
- Turn on oven and set temperature to 40C; wet humidifying paper
- Pretreatment
- Place slides on slide rack, add Pretreat 4 to entirely cover each section
- Incubate for 5 minutes at RT
- 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:
File:RNAScope Scoring Card.png
Buffer Prep
- 1X Wash Buffer
- Warm 50X wash buffer to 40C for 10-20 minutes before mixing
- Add 2.94L DDI water and 60 mL 50X wash buffer to make 3 L 1X wash buffer
- 1X wash buffer may be stored at RT for up to 1 month
- 50% Hematoxylin I
- Work in the fume hood
- Add 50% Hematoxylin by mixing hematoxylin I and DDI water
- Can be reused up to 1 week
- 0.02% Ammonia
- Add 1.43 mL of 1N ammonium hydroxide to 250 mL DI water; mix well 3-5 times
Do not reuse following reagents
- XYLENE
- 70% and 100% EtOH used in Dehydration during counterstain