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===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
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