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==Motor Neurons RNA FISH== *[[Matt:LabNotes/2015-6-8 | Previous Try]] *[[Matt:LabNotes/2015-6-15 | Continued from here (dye coupling)]] *Picked up second batch iPS derived motor neurons from Yeo Lab (Sebastian) **Fixed in 4% paraformaldehyde (fresh) at 3:00p 6/16/2015 ***RT for 15min **Permeabilized/store in 70% EtOH at 4C (~48 hours before RNA FISH experiment) ===RNA FISH Procedure=== *Using [[Matt:LabNotes/2015-6-15#Dye_Coupling | CUX2-488 KIT-594 CUX2-546 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 6.18.2015" **488 and 594 *Sample 2 labeled: "RNAFISH2 6.18.2015" **546 and 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 (2X volume) #*Hybridization Buffer 200ul #*Probe 10ul of each (5ul-488+5ul-594 and 5ul-546+5ul-647) #Wash the cells with 1ml of PBS (RNase free) 2 times #Add 1ml 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 37C #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=== *Olympus Imaging 6/19/2015 (Stored in DARTFISH-RNAFISH folder on desktop) **RNAFISH1: Alexa 488 (GFP filter) and Alexa 594 (TXRED filter) **RNAFISH2: Alexa 546 (Cy3 filter) and Alexa 647 (Cy5 filter) *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" *Still see a lot more background than example images on [https://plus.google.com/photos/108920949724677333478/albums/5859252817136626497?hl=en&partnerid=gplp0 Raj lab website] **[https://sites.google.com/site/singlemoleculernafish/faq#TOC-Imaging Arjun Raj FAQ for sm RNA FISH says to use cooled CCD camera] ====RNAFISH1==== *Pos1 488-CUX2 (GFP) **Most spots aren't in cell [[File:RNAFISH1_Pos1_GFP_StarSearch.png|450px]] *Pos1 594-KIT (TXRED) **Probably not mRNA signal? [[File:RNAFISH1_Pos1_TXRED_StarSearch.png|450px]] *Pos2 488-CUX2 (GFP) [[File:RNAFISH1_Pos2_GFP_StarSearch.png|450px]] *Pos2 594-KIT (TXRED) [[File:RNAFISH1_Pos2_TXRED_StarSearch.png|450px]] ====RNAFISH2==== *Pos1 546-CUX2 (Cy3) **Focus on dendrites [[File:RNAFISH2_Pos1_Dendrites_Cy3_StarSearch.png|450px]] **Focus on nucleus [[File:RNAFISH2_Pos1_Nucleus_Cy3_StarSearch.png|450px]] *Pos1 647-KIT (Cy5) **Focus on dendrites [[File:RNAFISH2_Pos1_Dendrites_Cy5_StarSearch.png|450px]] **Focus on nucleus [[File:RNAFISH2_Pos1_Nucleus_Cy5_StarSearch.png|450px]] *Pos2 546-CUX2 (Cy3) *Pos2 647-KIT (Cy5)
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