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==Experiment== ===PACT Protocol=== *[https://mgm.duke.edu/wp-content/uploads/2015/09/15-11-6-CLARITY-protocol.pdf Duke PACT protocol] #Fix embryo in 4% PFA at 37C for 15min #Make 4mL PACT solution '''on ice''' #*4% acrylamide in 1X PBS plus 0.25% VA-044 (degassed) #*3.584mL MilliQ H2O + 0.448mL 10X PBS + 0.448mL 40% acrylamide (cold) + 0.0112g 0.25% VA-044 (cold) #Wash with 1X PBS #Incubate embryo in solution at 4C overnight (20hrs) #Polymerize gel by incubating at 37C for 3hr #*Did not degas under Argon gas but can do next time if not polymerized enough #Make 500mL 8% SDS solution #*Could use 0.2 M boric acid or 0.01 M PBS (1X) #*6.183g boric acid, 200mL MilliQ H2O, pH with NaOH to pH=8.5, MilliQ H2O to 300mL total, then add 200mL 20% SDS #*50mL = 5mL 10X PBS + 20mL 20% SDS + 25mL H2O #Remove embryo from gel #*Pipette away surrounding hydrogel and wash with 1X PBS #Incubate in 8% SDS at 37C on rotator for overnight #*Tissue will not be fully clear until put into refractive index matching media #Wash embryo in PBS + 0.1% TX-100 at 37C on a rotator for 6 hrs #*Need to wash away SDS or it will precipitate forming cloudy spots ===CLARITY Protocol=== *[https://mgm.duke.edu/wp-content/uploads/2015/09/15-11-6-CLARITY-protocol.pdf Duke CLARITY protocol] #Make 4mL CLARITY solution '''on ice''' #*4% PFA, 0.95% acrylamide, 0.05% bis-acrylamide, 0.25% VA-044, 1X PBS (degassed) #*0.5mL 40% PFA + 0.125mL 40% 19:1 acrylamide:bis + 0.0125g VA-044 + 0.5mL 10X PBS + 3.875mL H2O #Incubate embryo in solution at 4C overnight (20hrs) #Polymerize gel by incubating at 37C for 3hr #Make 500mL 8% SDS solution #*Could use 0.2 M boric acid or 0.01 M PBS (1X) #*6.183g boric acid, 200mL MilliQ H2O, pH with NaOH to pH=8.5, MilliQ H2O to 300mL total, then add 200mL 20% SDS #*50mL = 5mL 10X PBS + 20mL 20% SDS + 25mL H2O #Remove embryo from gel #*Pipette away surrounding hydrogel and wash with 1X PBS #*Roll on kimwipe to remove bits of gel #Incubate in 8% SDS at 37C on rotator for overnight #*Tissue will not be fully clear until put into refractive index matching media #Wash embryo in PBS + 0.1% TX-100 at 37C on a rotator for 6 hrs #*Need to wash away SDS or it will precipitate forming cloudy spots ===FISSEQ=== *4 tdTomato positive embryos were taken by Paola for confocal/two-photon microscopy test *For tdTomato negative embryos do FISSEQ **4 CLARITY embryos and 2 PACT ===Day 1=== #Wash with PBS three times #Prepare 3X 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 |- | 4mM aa-dUTP||2 |- | 100uM FISSEQ_RT||5 |- | RNase Inhibitor||2 |- | M-MuLV RTase||10 |- | Total||200 |} #Add 100ul to each and incubate 10min at 4C and then ~16hr at 37C ===Day 2=== #Wash with 1X PBS once #Add 100ul cold BS(PEG)9 (12ul stock BS(PEG)9 + 588ul 1X PBS prepared on ice) to sample and incubate 1hr at RT #Wash with 1X PBS twice #Add 200ul 1M Tris (pH 8.0) for 30min at RT #Wash with 1X PBS twice #Add RNase H mix and incubate 1hr at 37C #*H2O - 168ul #*RNase H Buffer - 20ul #*Riboshredder - 2ul #*RNase H - 10ul #Wash with nuclease-free H2O twice #Add CircLigase mix and incubate 3hr at 60C #*H2O - 128ul #*CircLigase Buffer 10X - 20ul #*MnCl2 50mM - 10ul #*Betaine 5M - 40ul #*CircLigase II 100U/ul - 2ul #Wash with 1X PBS twice #*Lost many embryos due to sticking to pipette tip and unable to get off without scraping #*Only one CLARITY and one PACT embryo left #Add 200ul (1ul 200uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C #Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS #*PACT embryo stuck to pipette tip so cut off the tip and put in tube for incubation #Prepare RCA reaction mix '''on ice''' #*H2O - 174ul #*Phi29 Buffer 10X - 20ul #*dNTP 25mM - 2ul #*aa-dUTP 4mM - 2ul #*Phi29 DNA polymerase 100U/ul - 2ul #Add RCA mix and incubate at 30C overnight (~12hrs) ===Day 3=== #Wash with 1X PBS once #*Could not find embryo stuck to pipette tip #*Only one CLARITY embryo left #Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS prepared on ice) to sample and incubate 1hr at RT #Wash with 1X PBS twice #Add 200ul 1M Tris (pH 8.0) for 30min at RT #Wash with 1X PBS twice ====Imaging==== #Add 200ul of 0.5uM FISSEQ_Adpt Cy3 in 2X SSC + 30% formamide pre-heated to 75C #Incubate for 10min at RT #Wash with 2X SSC twice #Mount between coverslip and slide with standard mounting media and seal with nail polish *Essentially zero Cy3 signal in embryo
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