Daniel:Protocols/RNAScope

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Revision as of 16:06, 25 March 2015 by >Djacobse (→‎Chromogenic Staining Protocol)
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RNA Scope

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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

Chromogenic Staining Protocol

Tissue Prep (~30 minutes)

  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
  3. Incubate slides in 50% EtOH for 5 minutes at RT
  4. Incubate slides in 70% EtOH for 5 minutes at RT
  5. Incubate slides in 100% EtOH for 5 minutes at RT
  6. Incubate slides in fresh 100% EtOH for 5 minutes at RT
  7. Air dry on a paper towel for 5 minutes at RT
  8. Draw a hydrophobic barrier 2-4 times around each section with barrier pen.
    1. Let the barrier dry completely (~1 minute)
  9. Turn on oven at set temperature to 40C; wet humidifying paper
  10. Place slides on slide rack, add Pretreat 4 to entirely cover each section
  11. Incubate for 30 minutes at RT
    1. This time may be considerably shorter for different tissues, recommend 5 minutes for BA8
    2. Pretreat 3 is 1/2X Pretreat 4, can provide even gentler pretreat
  12. Take each slide from rack and tap or flick to remove excess liquid; immediately submerge in 1X PBS
  13. 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. Tap or flick to remove excess liquid from slides; place in slide rack; add ~4 drops of appropriate probe, cover the section
    1. 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
    1. Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down
  4. Tap or flick to remove excess liquid from slides; add ~4 drops of AMP1 to cover each section
    1. Incubate 30 minutes at 40C
  5. Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down
    1. Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down
  6. Tap or flick to remove excess liquid from slides; add ~4 drops of AMP2 to cover each section
    1. Incubate 15 minutes at 40C
  7. Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down
    1. Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down
  8. Tap or flick to remove excess liquid from slides; add ~4 drops of AMP3 to cover each section
    1. Incubate 30 minutes at 40C
  9. Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down
    1. Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down
  10. Tap or flick to remove excess liquid from slides; add ~4 drops of AMP4 to cover each section
    1. Incubate 15 minutes at 40C
  11. Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down
    1. Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down