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===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 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 [[Category:RNAFACS]] [[Category:RNAScope]] [[Category:PretreatII]]
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