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==RNA FISH Procedure== *Sample 1 labeled: "RNAFISH1 7.9.2015" **CUX2_odd-488 and CUX2_even-594 in Sebastian's neurons *Sample 2 labeled: "RNAFISH2 7.9.2015" **CUX2-488 only in Alex's neurons *Sample 3 labeled: "RNAFISH3 7.9.2015" **CUX2_odd-488 and CUX2_even-594 in Alex's neurons *Sample 4 labeled: "RNAFISH4 7.9.2015" **CUX2-488 and SNAP25-594 in Alex's neurons #Prepare 10ml Wash Buffer and let sit at RT #Thaw 500ul aliquot Hybridization Buffer and warm up to 37C #*Aliquots made by Dan #Prepare hybridization reaction #*Hybridization Buffer 100ul #*Probe 5ul for each dye-probe (final volume will be 105ul or 110ul) #Wash the cells with 1ml of PBS (RNase free) 2 times #Add 2ml Wash Buffer and let sit 10min at RT #Aspirate #Add 105ul or 110ul hybridization solution to each sample and incubate overnight at 37C (~22hrs) #Prepare 25ml Wash Buffer #Warm up 16ml Wash Buffer and 8ml Wash Buffer with DAPI to 37C #Rinse with 2ml Wash Buffer #Add 2ml Wash Buffer and incubate 30min at 37C #Replace with 2ml Wash Buffer with DAPI and incubate 30min at 30C #Wash with 2X SSC (warmed to 30C) twice #Add 2X SSC ====Buffer Prep==== *Wash Buffer **20X SSC 5mL **Formamide 5mL **RNase free H2O 40ml *Wash Buffer with DAPI **Wash Buffer 10ml **DAPI (100ug/ml) 50ul ***Stored in brown opaque tube in 4C *Hybridization Buffer **RNAse free water 5.3 mL **SSC 20X 1 mL **Dextran sulfate 2 mL **Formamide 1 mL **E coli tRNA 500 uL **RVC 200 mM (warm to 37) 100 uL **BSA 50 mg/mL 40 uL
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