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===RNA FISH=== *Using [[Matt:LabNotes/2015-6-5#Dye_Coupling | SLC6A1-488 SATB2-594 Probes]] *[[Daniel:Notebook/RNAFISH/2015-3-4 | Dan's best practice FISH protocol]] *[[Daniel:Protocols/TissueFISH#Linnarsson_Lab_Protocol | Linnarsson complete FISH protocol]] *Sample: RT 15min **Labeled "RNAFISH1 6.8.2015" *Sample: 37C 10min **Labeled "RNAFISH2 6.8.2015" #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 (2X volume) #*Hybridization Buffer 200ul #*Probe 10ul of each (488 and 594) #Wash the cells with 1ml of PBS (RNase free) 2 times #Add Wash Buffer and let sit 10min at RT #Aspirate #Add 110ul hybridization solution to each sample and incubate overnight at 37C (~18hrs) #Warm up 6ml Wash Buffer and 2ml Wash Buffer with DAPI to 37C #Rinse with 2ml Wash Buffer #Add 1ml Wash Buffer and incubate 30min at 37C #Replace with 1ml 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 ====Results==== *Tried imaging with confocal using 488 and 552 lasers **Only saw autofluorescence from cell, looked really cool to see all the dendrites/axons branching from cells but no spots *[https://sites.google.com/site/singlemoleculernafish/faq#TOC-Imaging Arjun Raj FAQ for sm RNA FISH suggests using epifluorescence is better than confocal] *Used Olympus **GFP (for Alexa 488) and Texas Red (for Alexa 594) filter cubes ***300ms-1000ms exposure **DAPI ***~20ms exposure *Merged the different channels together (blue=DAPI,green=GFP,red=TXRED,grey=BF) **Green and red overlap everywhere (only yellow can be seen) therefore no specific signal ***Saved in DARTFISH-RNAFISH folder on Desktop (6/10/2015) ***Will try higher expressed genes (KIT and CUX2): [[Matt:LabNotes/2015-6-18#RNA_FISH | Try #2]] [[File:20150610_MotorNeur_RNAFISH2_Dish5_Pos1_composite.jpg|500px]]
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