Daniel:Notebook/RNAFACS/2015-10-21

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Revision as of 17:08, 21 October 2015 by >Djacobse
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RNA Scope Trial Run 4: Pretreat

Back to Calender

So after talking with acdbio people yesterday, they recommended that I reduce the stringency of my pretreat. I did have success over the weekend with the pretreat as usual, but there were only a few nuclei to look at, which could be the result of several things:

  • Over-pretreating
  • Bad adhesion (shouldn't be a problem, since other times had loads of nuclei)
  • Low nuclei count in Blue's sort samples (possible, but not much I can do on that one)

So long story short, we'll give it another go with lower amount of pretreat.

Samples: From Andrew: Cerebellum, May 2014, Patient ######

Nuclei Adhesion

  1. Clean glass slide in 100% EtOH; allow to air dry
    1. Draw a square around the desired adhesion area using a hydrophobic barrier pen
  2. Remove cells from -80C
  3. Thaw, and add 500uL of 4% paraformaldehyde to the tube and wait 2 min
  4. Centrifuge cells for 5 min at 1000g
    1. Remove supernatant, careful not to disturb the pellet
  5. Add 500 uL of 70% EtOH
  6. Centrifuge cells for 5 min at 1000g
    1. Remove supernatant, careful not to disturb the pellet
  7. Resuspend nuclei in 20 uL 70% EtOH
  8. Deposit on to glass slide
  9. Use a coverslip to gently spread the 20 uL across the slide
    1. Be careful not to scratch the slide
  10. Dry the slide using a heating block at 60C for 10-15 min

RNA Scope

Sample Matrix

  Slide 1 Slide 2
C1 RBFOX3/NeuN +Control
C2 -Control +Control
C3 FEZF2 +Control

Nuclei Prep

  1. Rehydration
    1. Incubate slides in 70% EtOH for 2 minutes
    2. Incubate slides in 50% EtOH for 2 minutes
    3. Incubate slides in 1X PBS for 10 minutes
    4. May want to retrace hydrophobic barrier; can skip
  2. Apply Pretreat 3
    1. Dilute pretreat 3 1:15; 1 drop of pretreat 3: ~200 uL 1X PBS
    2. Remove excess liquid; add 150 uL drops of diluted pretreat 3
    3. Incubate for 10 min at RT (in oven)
    4. Flick to remove excess liquid
    5. Rinse in fresh 1X PBS; be sure to agitate to clean well

Fluorescent Assay

  1. Prepare-Reagents
    1. Prepare probes by warming to 40C for 10 minutes, then cool to RT
    2. Briefly spin down C2 probes to collect the liquid
    3. Combine C2 and C1 probes by pipetting into a new 1.5mL tube at 1:50 ratio
    4. Prepare 1X Wash Buffer
    5. Place AMP1-4 FL reagents at RT
    6. Ensure HybEZ OVEN and Control Tray are at 40C
  2. Hybridize Probe
    1. Flick to remove excess liquid; add 2-4 drops of probe
    2. Incubate for 2 hrs at 40C
    3. Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
    4. Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
  3. AMP1-FL
    1. Flick to remove excess liquid
    2. Add 2-4 drops of AMP1-FL; Incubate for 30 minutes at 40C
    3. Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
    4. Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
  4. AMP2-FL
    1. Flick to remove excess liquid
    2. Add 2-4 drops of AMP2-FL; Incubate for 15 minutes at 40C
    3. Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
    4. Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
  5. AMP3-FL
    1. Flick to remove excess liquid
    2. Add 2-4 drops of AMP3-FL; Incubate for 30 minutes at 40C
    3. Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
    4. Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
  6. AMP4-FL
    1. Flick to remove excess liquid
    2. Add 2-4 drops of AMP4-FL ALT C; Incubate for 15 minutes at 40C
    3. Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
    4. Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
  7. Counterstain and Mount
    1. Flick to remove excess liquid;
    2. Add 2-4 drops DAPI; Incubate 30 sec at RT
    3. Remove DAPI and immediate place 1-2 drops of fluorescent mounting medium
    4. Seal with a coverslip; avoid bubbles
  8. Store in the dark at 4C
  9. Recommended Viewing at 20-40X magnification