Daniel:Notebook/RNAFISH/2015-3-26: Difference between revisions

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>Djacobse
>Djacobse
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##Incubate slides in fresh 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
##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)
##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
#Turn on oven at set temperature to 40C; wet humidifying paper
#Pretreatment
#Pretreatment

Revision as of 17:47, 26 March 2015

RNA Scope Chromogenic

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Tissue Prep (~30 minutes)

  1. Fixation
    1. Chill 4 %PFA (Fresh 4% PFA or 10% NBF in 1X PBS) to 4C (ice)
    2. Remove slides from -80C and immediately immerse in pre-chilled fixative for 15 minutes
  2. Dehydration
    1. Incubate slides in 50% EtOH for 5 minutes at RT
    2. Incubate slides in 70% EtOH for 5 minutes at RT
    3. Incubate slides in 100% EtOH for 5 minutes at RT
    4. Incubate slides in fresh 100% EtOH for 5 minutes at RT
    5. Air dry on a paper towel for 5 minutes at RT
  3. Draw a hydrophobic barrier 2-4 times around each section with barrier pen; Let the barrier dry completely (~1 minute)
  4. Turn on oven at set temperature to 40C; wet humidifying paper
  5. Pretreatment
    1. Place slides on slide rack, add Pretreat 4 to entirely cover each section
    2. Incubate for 5 minutes at RT
    3. Take each slide from rack and tap or flick to remove excess liquid; immediately submerge in 1X PBS
    4. Wash slides in 1X PBS by moving rack up and down 3-5 times; repeat with fresh 1X PBS

RNA Scope Assay

  1. Place AMP 1-6 reagents at RT; Warm oven to 40C; warm probes for 10 minutes at 40C, swirl gently to mix
  2. Probe Hybridization
    1. Tap or flick to remove excess liquid from slides; place in slide rack; add ~4 drops of appropriate probe, cover the section
    2. Incubate for (at least) 2 hours at 40C
    3. Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down
    4. Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down
  3. AMP1
    1. Tap or flick to remove excess liquid from slides; add ~4 drops of AMP1 to cover each section
    2. Incubate 30 minutes at 40C
    3. Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down
    4. Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down
  4. AMP2
    1. Tap or flick to remove excess liquid from slides; add ~4 drops of AMP2 to cover each section
    2. Incubate 15 minutes at 40C
    3. Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down
    4. Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down
  5. AMP3
    1. Tap or flick to remove excess liquid from slides; add ~4 drops of AMP3 to cover each section
    2. Incubate 30 minutes at 40C
    3. Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down
    4. Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down
  6. AMP4
    1. Tap or flick to remove excess liquid from slides; add ~4 drops of AMP4 to cover each section
    2. Incubate 15 minutes at 40C
    3. Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down
    4. Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down
  7. AMP5
    1. Tap or flick to remove excess liquid from slides; add ~4 drops of AMP5 to cover each section
    2. Incubate 30 minutes at RT
    3. Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down
    4. Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down
  8. AMP6
    1. Tap or flick to remove excess liquid from slides; add ~4 drops of AMP3 to cover each section
    2. Incubate 15 minutes at RT
    3. Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down
    4. Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down
  9. Signal Detection
    1. 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
    2. Tap or flick to remove excess liquid from slides; place ~120 uL DAB onto each section
    3. Incubate 10 minutes at RT
    4. Submerge slides in DDI water; agitate 3-5 times; replace with fresh DDI water
  10. Counterstain
    1. Submerge slides in 50% hematoxylin I solution for 2 minutes at RT; slides will be purple
    2. Immediately transfer slides back to DDI water; agitate 3-5 times; repeat with fresh DDI water until slides are clear and sections are purple
    3. Replace DDI water with 0.02% ammonia water; agitate 2-3 times; sections should turn blue
    4. Submerge slides in DDI water, wash 3-5 times
  11. Dehydrate
    1. Submerge slides in 70% EtOH in fume hood for 2 minutes with occasional agitation
    2. Submerge slides in 100% EtOH in fume hood for 2 minutes with occasional agitation
    3. Submerge slides in fresh 100% EtOH in fume hood for 2 minutes with occasional agitation
    4. Submerge slides in XYLENE for 5 minutes in fume hood with occasional agitation
  12. Mounting
    1. Remove slides from xylene and lay flat with sections up in the fume hood
    2. Mount slides by adding 1-2 drops of cytoseal mounting medium and seal with a coverslip; avoid air bubbles
    3. Air dry slides for at least 5 minutes
  13. Evaluation
    1. Evaluate under standard brightfield microscope at 20-40X magnification
    2. Positive control signal should be visible as punctate dots within cell nucleii at 20-40X magnification
    3. Negative control may have signal; One dot to every 10 cells is acceptable
    4. Scoring:

File:RNAScope Scoring Card.png

Buffer Prep

  1. 1X Wash Buffer
    1. Warm 50X wash buffer to 40C for 10-20 minutes before mixing
    2. Add 2.94L DDI water and 60 mL 50X wash buffer to make 3 L 1X wash buffer
    3. 1X wash buffer may be stored at RT for up to 1 month
  2. 50% Hematoxylin I
    1. Work in the fume hood
    2. Add 50% Hematoxylin by mixing hematoxylin I and DDI water
    3. Can be reused up to 1 week
  3. 0.02% Ammonia
  4. Add 1.43 mL of 1N ammonium hydroxide to 250 mL DI water; mix well 3-5 times

Do not reuse following reagents

  1. XYLENE
  2. 70% and 100% EtOH used in Dehydration during counterstain