Daniel:Notebook/RNAFACS/2015-10-22: Difference between revisions
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>Djacobse (Created page with "=RNA Scope Trial Run 5: Pretreat II= Back to Calender Yesterday's trial did not go so well. I think Andre...") |
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#Use a coverslip to gently spread the 20 uL across the slide | #Use a coverslip to gently spread the 20 uL across the slide | ||
##Be careful not to scratch the slide | ##Be careful not to scratch the slide | ||
#Dry the slide using a heating block at 60C for 10 | #Dry the slide using a heating block at 60C for 10 min | ||
==RNA Scope== | ==RNA Scope== | ||
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##May want to retrace hydrophobic barrier; can skip | ##May want to retrace hydrophobic barrier; can skip | ||
#Apply Pretreat 3 | #Apply Pretreat 3 | ||
##'''Dilute pretreat 3 1:15; 1 drop of pretreat 3: ~ | ##'''Dilute pretreat 3 1:15; 1 drop of pretreat 3: ~30uL; add 420 uL 1X PBS to 1 drop pretreat 3''' | ||
##Remove excess liquid; add ''' | ##Remove excess liquid; add '''120 uL drops of diluted pretreat 3''' | ||
##Incubate for 10 min at RT (in oven) | ##Incubate for 10 min at RT (in oven) | ||
##Flick to remove excess liquid | ##Flick to remove excess liquid | ||
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#Store in the dark at 4C | #Store in the dark at 4C | ||
#Recommended Viewing at 20-40X magnification | #Recommended Viewing at 20-40X magnification | ||
[[Category:RNAFACS]] [[Category:RNAScope]] [[Category:PretreatII]] |
Latest revision as of 17:13, 22 October 2015
RNA Scope Trial Run 5: Pretreat II[edit]
Yesterday's trial did not go so well. I think Andrew's samples were too degraded. However, Blue gave me some samples that are more relevant. These samples should not only be NeuN+, but they should also have FEZF2.
Samples: From Blue:
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
- Thaw, and add 500uL of 4% paraformaldehyde to the tube and wait 2 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 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
- Dilute pretreat 3 1:15; 1 drop of pretreat 3: ~30uL; add 420 uL 1X PBS to 1 drop pretreat 3
- Remove excess liquid; add 120 uL drops of diluted 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 C; 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