Daniel:Notebook/RNAFACS/2015-10-7: Difference between revisions
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>Djacobse (Created page with "=RNA Scope Trial Run= Back to Calendar Now that I know that the nuclear adhesion protocol works its time to test out RNA Scope (fluorescent), inc...") |
>Djacobse |
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==RNA Scope== | ==RNA Scope== | ||
'''Sample Matrix''' | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="font-size:12pt" | |||
| align="center" width="65" height="15" valign="bottom" | | |||
|style="background-color:#CCC0DA;font-weight:bold" width="94" align="center" valign="bottom" | Slide 1 | |||
|style="background-color:#CCC0DA;font-weight:bold" width="65" align="center" valign="bottom" | Slide 2 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | C1 | |||
| valign="bottom" | RBFOX3/NeuN | |||
| align="center" valign="bottom" | +Control | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| height="15" valign="bottom" | C2 | |||
| valign="bottom" | -Control | |||
| align="center" valign="bottom" | +Control | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | C3 | |||
| valign="bottom" | FEZF2 | |||
| align="center" valign="bottom" | +Control | |||
|} | |||
===Nuclei Prep=== | ===Nuclei Prep=== |
Latest revision as of 18:53, 7 October 2015
RNA Scope Trial Run[edit]
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[edit]
- 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[edit]
Sample Matrix
Slide 1 | Slide 2 | |
C1 | RBFOX3/NeuN | +Control |
C2 | -Control | +Control |
C3 | FEZF2 | +Control |
Nuclei Prep[edit]
- 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[edit]
- 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