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=BA8 Section DARTFISH with Fiducial Beads= *Try again: [[Matt:LabNotes/2015-8-3]] **with fresh PFA *Practice DARTFISH on BA8 tissue sections *Use FluoSpheres Amine-Modified Microspheres 0.2um (505/515) as fiducial markers ==DARTFISH on BA8== #Prepare plastic culture dish with hole, tweezers, large plastic dish, and 50C hot plate #*EtOH sterilize and UV #Made 40ml 4% PFA #*10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O #Take out BA8 section from -80C and incubate on 50C hot plate for 3min #Submerge section in 5ml 4% PFA in 6 well culture plate for 15min at 37C #Wash with 2X SSPE by submerging in 2 clean wells of culture plate with 5ml SSPE #Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive #*Still had leak so used gap-filling adhesive 200 to seal #Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT #*0.25% TX-100 in 2XSSPE tube had crack in cap so not completely sealed #Wash with nuclease-free H2O three times #Add 0.01% Pepsin in 0.1N HCl and incubate for 5min at 37C #*2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O #Wash with nuclease-free PBS three times #*Can already see tissue is degrading #Prepare Reverse Transcription Mix '''on ice''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H2O||157 |- | 10X M-MuLV Buffer||20 |- | 25mM dNTP||2 |- | 2mM aa-dUTP||4 |- | 100uM N9||5 |- | RNase Inhibitor||2 |- | M-MuLV RTase||10 |- | Total||200 |} #Incubate 10min at 4C and then ~15hr at 37C #*Tissue is slightly degraded but mostly okay #Wash with 1X PBS once #Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 1X PBS) and incubate 1hr at RT #Wash with 1X PBS twice #Add Tris pH8.0 and incubate 30min at RT #Wash with 1X PBS twice #RNA Removal #*Add RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C #Wash with H2O twice #Add 66nM Ampligase Mix (no suppressor) and incubate ~3hr at 60C as oven slowly decreases to 55C and held for another ~16hr #*Wrapped with parafilm to prevent evaporation (works very well) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | DEPC-H2O||121 |- | Ampligase Buffer||20 |- | [[Matt:JustinLabNotes/2015-7-9#Qubit_Quantification | Padlock Probes 337nM]]||39 |- | Ampligase||20 |- | Total||200 |} #Wash with 1X PBS once #Add 1uM FISSEQ_ppRCA (0.5ul 200uM FISSEQ_ppRCA + 199.5ul 2X SSC + 30% formamide) and incubate 1hr at 55C #*Preheated to 65C before adding #Wash with 2X SSC once, 1X SSC once, and 1X PBS once #Add RCA mix and incubate 6hr at 30C {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H2O||172 |- | 10X Phi29 Buffer||20 |- | 25mM dNTP||2 |- | 2mM aa-dUTP||4 |- | Phi 29 (low conc)||2 |- | Total||200 |} #Wash with 10mM HEPES #Dilute beads 1:500 in 10mM HEPES (1ul stock beads + 249ul H2O + 250ul 20mM HEPES pH 7.3) #*Make 1.2ml aliquots of stock beads and store in dcProbe box in 4C #Sonicate for 3 minutes with microtip probe in 1.5ml tube #*Power = 3, 3sec on 2sec off #Add to sample and incubate 5min at RT #Wash with 10mM HEPES once #Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 20mM HEPES) and incubate at RT for 1hr #Wash with 1X PBS twice #Add 1M Tris pH 8.0 and incubate at RT for 30min #Wash with 1X PBS twice and store in 1X PBS at 4C overnight ===Results=== ====Pos1: Gain 550==== *Fluorescent beads saturated intensity *All Rolonies [[File:MAX_20150804_BA8DARTFISH_FidBeads_Gain550_Pos1_ch00.jpg|650px]] *Fidicual Beads [[File:MAX_20150804_BA8DARTFISH_FidBeads_Gain550_Pos1_ch01.jpg|650px]] ====Pos2: Gain 400==== *Decreased gain of 488 channel *All Rolonies [[File:MAX_20150804_BA8DARTFISH_FidBeads_Gain400_Pos2_ch00.jpg|650px]] *Fidicual Beads [[File:MAX_20150804_BA8DARTFISH_FidBeads_Gain400_Pos2_ch01.jpg|650px]]
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