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===RNA Scope Assay=== #Place AMP 1-6 reagents at RT; Warm oven to 40C; warm probes for 10 minutes at 40C, swirl gently to mix #Probe Hybridization ##Tap or flick to remove excess liquid from slides; place in slide rack; add ~4 drops of appropriate probe, cover the section ##Incubate for (at least) 2 hours at 40C ##Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down ##Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down #AMP1 ##Tap or flick to remove excess liquid from slides; add ~4 drops of '''AMP1''' to cover each section ##Incubate 30 minutes at 40C ##Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down ##Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down #AMP2 ##Tap or flick to remove excess liquid from slides; add ~4 drops of '''AMP2''' to cover each section ##Incubate 15 minutes at 40C ##Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down ##Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down #AMP3 ##Tap or flick to remove excess liquid from slides; add ~4 drops of '''AMP3''' to cover each section ##Incubate 30 minutes at 40C ##Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down ##Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down #AMP4 ##Tap or flick to remove excess liquid from slides; add ~4 drops of '''AMP4''' to cover each section ##Incubate 15 minutes at 40C ##Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down ##Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down #AMP5 ##Tap or flick to remove excess liquid from slides; add ~4 drops of '''AMP5''' to cover each section ##Incubate 30 minutes at RT ##Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down ##Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down #AMP6 ##Tap or flick to remove excess liquid from slides; add ~4 drops of '''AMP3''' to cover each section ##Incubate 15 minutes at RT ##Remove tray; remove excess liquid from each slide and submerge in 1X Wash buffer for 2 minutes, agitate by moving slides up and down ##Submerge in fresh 1X wash buffer for 2 minutes, agitate by moving slides up and down #Signal Detection ##Mix equal volumes of Brown-A and Brown-B (DAB substrate) (e.g. 2 drops each); make ~120 uL DAB per section, mix well 3-5 times ##Tap or flick to remove excess liquid from slides; place ~120 uL DAB onto each section ##Incubate 10 minutes at RT ##Submerge slides in DDI water; agitate 3-5 times; replace with fresh DDI water #Counterstain ##Submerge slides in 50% hematoxylin I solution for 2 minutes at RT; slides will be purple ##Immediately transfer slides back to DDI water; agitate 3-5 times; repeat with fresh DDI water until slides are clear and sections are purple ##Replace DDI water with 0.02% ammonia water; agitate 2-3 times; sections should turn blue ##Submerge slides in DDI water, wash 3-5 times #Dehydrate ##Submerge slides in 70% EtOH in fume hood for 2 minutes with occasional agitation ##Submerge slides in 100% EtOH in fume hood for 2 minutes with occasional agitation ##Submerge slides in fresh 100% EtOH in fume hood for 2 minutes with occasional agitation ##Submerge slides in XYLENE for 5 minutes in fume hood with occasional agitation #Mounting ##Remove slides from xylene and lay flat with sections up in the fume hood ##Mount slides by adding 1-2 drops of cytoseal mounting medium and seal with a coverslip; avoid air bubbles ##Air dry slides for at least 5 minutes #Evaluation ##Evaluate under standard brightfield microscope at 20-40X magnification ##Positive control signal should be visible as punctate dots within cell nucleii at 20-40X magnification ##Negative control may have signal; One dot to every 10 cells is acceptable ##Scoring: [[Image:RNAScope_Scoring_Card.png|600px]]
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