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==Protocol== ===Day 1=== #Prepare hydrogel mix ##Positive Control: [[Matt:LabNotes/2016-2-9#Experiment|Standard hydrogel]] + 4ul of PKP2 beads ##5% PA Gel: [[Matt:LabNotes/2016-2-9#Experiment|Standard hydrogel]] ##5% PA Gel + 4% PFA: [[Matt:LabNotes/2016-2-9#Experiment|Standard hydrogel]] + 4% PFA #Attach 3 ~120um thick adhesives to 3 Vectabond + Bind-silane treated coverslips #Add 6ul PKP2 beads diluted in 50ul H2O to coverslips right on top of 2 magnets #*Pipette away extra H2O leaving only dried beads #Inject ~50ul of 200ul hydrogel mix into each dish (underneath another coverslip) #Vacuum seal bag and then pump with Argon gas #Let set at RT for 30min #Wash with nf-H2O twice #*5% PA Gel + 4% PFA failed to polymerize *Gels are sitting in 1X PBS at 4C #Prepare Ampligase mix on ice #*Preheat H2O + Padlock probes to 85C and then snap cool before adding Ampligase buffer and enzyme {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | DEPC-H2O||70 |- | Ampligase Buffer||10 |- | PKP2_controlPP 10nM||10 |- | Ampligase||10 |- | Total||100 |} #Add mix to sample and incubate for 30min at 37C #Move sample to 60C and oven slowly decreases to 55C and held for ~24hr ===Day 2=== #Wash with 1X PBS once #Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C #*Preheated to 70C before adding #Wash with 2X SSC once, 1X SSC once, and 1X PBS once #Add RCA mix and incubate 15hr at 30C {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H2O||174 |- | 10X Phi29 Buffer||20 |- | 25mM dNTP||2 |- | 4mM aa-dUTP||2 |- | Phi 29 (low conc)||2 |- | Total||200 |} ===Day 3=== #Wash with 1X PBS twice #Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) for 1hr at RT #Wash with 1X PBS twice #Add 1M Tris ph8.0 for 30min at RT
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