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=Motor Neurons RNA FISH with cooled CCD= *Photometric QUANTEM Camera **Cooled to -30C **100X objective *Ex/Em Filters available [[File:IMG_0517.JPG | 450px]] *Alexa488 **495/519 (FITC equivalent) **Filter 5/Filter 2 *Alexa564 **556/573 (tetramethylrhodamine) **No filters available *Alexa594 **590/617 (Texas Red) **Filter 8/Filter 5 *Alexa647 **647/665 (Cy5) **Filter 4/Filter 6-7 *[[Matt:LabNotes/2015-6-8 | First Try]] *[[Matt:LabNotes/2015-6-18 | Second Try]] *[[Matt:LabNotes/2015-6-15 | Probe dye coupling]] *[[Matt:LabNotes/2015-6-18 | Second batch iPS derived motor neurons from Yeo Lab (Sebastian)]] **Permeabilized/stored in 70% EtOH at 4C (2 weeks before RNA FISH experiment) ===RNA FISH Procedure=== *Using [[Matt:LabNotes/2015-6-15#Dye_Coupling | CUX2-488 KIT-594 KIT-647]] *[[Daniel:Notebook/RNAFISH/2015-3-4 | Dan's best practice FISH protocol]] *[[Daniel:Protocols/TissueFISH#Linnarsson_Lab_Protocol | Linnarsson complete FISH protocol]] *Sample 1 labeled: "RNAFISH1 6.30.2015" **488 *Sample 2 labeled: "RNAFISH2 6.30.2015" **594 *Sample 3 labeled: "RNAFISH3 6.30.2015" **647 #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 #Wash the cells with 1ml of PBS (RNase free) 2 times #Add 2ml Wash Buffer and let sit 10min at RT #Aspirate #Add 105ul hybridization solution to each sample and incubate overnight at 37C (~23hrs) #Prepare 15ml Wash Buffer #Warm up 12ml Wash Buffer and 3ml 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 ===Results=== *Only CUX2 (488) probes worked **My guess is CUX2 is most highly expressed in these cells **To confirm next time I can use CUX2 probes labeled with 2 different dyes ***Odd probes labeled one color and even probes labeled the other, should see colocalization of 2 colors ====Raw Images==== *RNAFISH1_Pos1 **Exposure only 1sec vs 5sec for other images [[File:2_w1Alexa488.jpg|350px]] [[File:2_w2DIC.jpg|350px]] *RNAFISH1_Pos2 [[File:3_w1Alexa488.jpg|350px]] [[File:3_w2DIC.jpg|350px]] *RNAFISH1_Pos3 [[File:5_w1Alexa488.jpg|350px]] [[File:5_w2DIC.jpg|350px]] *RNAFISH1_Pos4 [[File:7_w1Alexa488.jpg|350px]] [[File:7_w2DIC.jpg|350px]] *RNAFISH3_Pos1 with 488 excitation (use exact same microscope settings as RNAFISH1 for '''Negative Control''') [[File:Dish3_5_w1Alexa488.jpg|350px]] [[File:Dish3_5_w2DIC.jpg|350px]] *RNAFISH3_Pos2 with 488 excitation (use exact same microscope settings as RNAFISH1 for '''Negative Control''') [[File:Dish3_6_w1Alexa488.jpg|350px]] [[File:Dish3_6_w2DIC.jpg|350px]] ====StarSearch==== *Use [http://rajlab.seas.upenn.edu/StarSearch/launch.html StarSearch from Raj Lab] **Doesn't work in Chrome browser (use IE) **Uses a histogram of pixel intensities and a threshold to determine how many "stars" *RNAFISH1_Pos2 [[File:3_w1Alexa488_StarSearch.png]] *RNAFISH1_Pos3 [[File:5_w1Alexa488_StarSearch.png]] *RNAFISH1_Pos4 [[File:7_w1Alexa488_StarSearch.png]] *RNAFISH3_Pos1 with 488 excitation (use exact same microscope settings as RNAFISH1 for '''Negative Control''') **Can't process image, something about StarSearch algorithm makes image almost all white/saturated *RNAFISH3_Pos2 with 488 excitation (use exact same microscope settings as RNAFISH1 for '''Negative Control''') **Identified 6 spots but you can tell they're not real distinct spots [[File:Dish3_6_w1Alexa488_StarSearch.png]]
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