Daniel:Notebook/RNAFACS/2015-10-7
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RNA Scope Trial Run
Now that I know that the nuclear adhesion protocol works its time to test out RNA Scope (fluorescent), including trying out the Huang lab microscope.
Nuclei Adhesion
- Clean glass slide in 100% EtOH; allow to air dry
- Draw a square around the desired adhesion area using a hydrophobic barrier pen
- Remove cells from -80C
- Separate into two tubes (15 uL ea) and spin down for 5 min at 1000g
- Remove supernatant, careful not to disturb the pellet
- Add 500uL of 4% paraformaldehyde to the tube and wait 5 min
- Centrifuge cells for 5 min at 1000g
- Remove supernatant, careful not to disturb the pellet
- Add 500 uL of 70% EtOH
- Centrifuge cells for 5 min at 1000g
- Remove supernatant, careful not to disturb the pellet
- Resuspend nuclei in 20 uL 70% EtOH
- Deposit on to glass slide
- Use a coverslip to gently spread the 20 uL across the slide
- Be careful not to scratch the slide
- Dry the slide using a heating block at 60C for 10-15 min
RNA Scope
Since I don't have one gene yet, both samples will be RBFOX3/NeuN (C1) FEZF2 (C2).
Nuclei Prep
- Rehydration
- Incubate slides in 70% EtOH for 2 minutes
- Incubate slides in 50% EtOH for 2 minutes
- Incubate slides in 1X PBS for 10 minutes
- May want to retrace hydrophobic barrier; can skip
- Apply Pretreat 3
- Remove excess liquid; add 2-4 drops of pretreat 3
- Incubate for 10 min at RT (in oven)
- Flick to remove excess liquid
- Rinse in fresh 1X PBS; be sure to agitate to clean well
Fluorescent Assay
- Prepare-Reagents
- Prepare probes by warming to 40C for 10 minutes, then cool to RT
- Briefly spin down C2 probes to collect the liquid
- Combine C2 and C1 probes by pipetting into a new 1.5mL tube at 1:50 ratio
- Prepare 1X Wash Buffer
- Place AMP1-4 FL reagents at RT
- Ensure HybEZ OVEN and Control Tray are at 40C
- Hybridize Probe
- Flick to remove excess liquid; add 2-4 drops of probe
- Incubate for 2 hrs at 40C
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- AMP1-FL
- Flick to remove excess liquid
- Add 2-4 drops of AMP1-FL; Incubate for 30 minutes at 40C
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- AMP2-FL
- Flick to remove excess liquid
- Add 2-4 drops of AMP2-FL; Incubate for 15 minutes at 40C
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- AMP3-FL
- Flick to remove excess liquid
- Add 2-4 drops of AMP3-FL; Incubate for 30 minutes at 40C
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- AMP4-FL
- Flick to remove excess liquid
- Add 2-4 drops of AMP4-FL ALT B; Incubate for 15 minutes at 40C
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes
- Counterstain and Mount
- Flick to remove excess liquid;
- Add 2-4 drops DAPI; Incubate 30 sec at RT
- Remove DAPI and immediate place 1-2 drops of fluorescent mounting medium
- Seal with a coverslip; avoid bubbles
- Store in the dark at 4C
- Recommended Viewing at 20-40X magnification