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==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=== #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=== #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 50 uL Draq5; Incubate 1 min 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 [[Category:RNAFACS]] [[Category:RNAScope]] [[Category:RNAScope-10X-II]]
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