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====Procedure==== =====Fixation===== *Process *#4% Formaldehyde : 2.5mL 16% Paraformaldehyde + 1mL 10x DEPC-PBS + 6.5mL DEPC-H2O (Tot. 10mL) *#*16% Paraformaldehyde Solution) *#*Manufacture : Electron Microscopy Diatome --> 16% PARAFORMALDEHYDE AQ SOLUTN 50-980-486, No.:15700 *#*Supply : (Fisher Scientific), 50980486 *#*Paraformaldehyde - methanol free solution. A more efficient and rapid penetrant fixative used in combination with Glutaraldehyde and Acrolein fixatives. Easy to break, prescored, 10 ml ampoules sealed under inert gas 10 ml *#Take Tissue glass slide from -80C, and put them to room temp. for 2min. *#Add 4% formamide on a tissue, incubate for 30min at room temp. *#Wash with PBS 3 times (add PBS in 3 wells of 6 well plate and series washing with glasses) *#Attach glass with glue *#*Cyanoacrylate adhesives : High-viscosity, Sigma-Aldrich, Z105902-1EA *#Wipe PBS from the glass (around tissue) *#Apply glue along the edge of the glass, put on the bottom of MatTek dish, wait for 5min [[File:TissueGlassOnMatTek_2.png|550px]] [[File:TissueGlassOnMatTek_1.png|400px]] =====1st Rolony Process===== *RT *#Wash with H2O for 5min. twice *#Add 0.25% TX-100 + DEPC-H2O, incubate for 15min. at room temp. *#Wash with DEPC-H2O twice *#Add 200 ul 0.1% pepsin in 0.1 N HCl (4uL of 5% Pepsin + 10uL of 2N HCl + 186uL DEPC-H2O), incubate for 10 min at 37C *#Wash with DEPC-PBS three times to inactivate pepsin *#Add RT mix (Hexamer, -a-dUTP) *#*DEPC-H2O 161uL *#*M-MuLV Buffer 20uL *#*25mM dNTP 2uL *#*100uM random hexamer RT Primer 5uL *#*Rnase Inhibitor 2uL *#*M-MuLV reverse transcriptase 10 uL *#Incubate the specimen with the reaction mixture for 10 min at 4°C, then transfer to 37°C for overnight. *Circligase II *#Wash with PBS once, skip BS(PEG)9 *#Add RNase H mix, incubate for 1hr. at 37C *#*H2O 168uL *#*Riboshredder 2uL *#*RNase H buffer 20uL *#*RNase H 10uL *#Wash with nucleaus-free H2O twice *#Add CircLigase II mix, incubate at 60C for 2hr. *#*H2O 128uL *#*Buffer 20uL *#*50mM MnCl2 10uL *#*5M Betaine 40uL *#*CircLigaseII 2uL *RCA *#Add 0.5uM RCA Primer in 200uL (2x SSC + 30% Formamide), incubate at 60C for 1hr. *#*1uL of 100uM RCA Primer + 199uL (2X SSC + 30% Formamide) *#Wash with 2x SSC, 1x SSC twice each *#Prepare RCA reaction mix *#*H2O 172uL *#*Buffer 20uL *#*25mM dNTP 2uL *#*2mM a-dUTP 4uL *#*Phi29(100 U/µl) 2uL *#Add RCA mix, incubate at 30C for overnight *Post fix *#Wash with PBS once *#add 200uL BS(PEG)9 mix (4uL BS(PEG)9 in 196uL 1x PBS), incubate for 1hr at RT *#Wash with PBS once, add Tris pH 8.0, incubate for 30min *#Wash with PBS once *Imaging *#Prepare detection Probe : Final Conc. 1uM --> 2uL of 100uM Cy3-Adapt + 198uL (2xSSC + 30% Formamide) *#Preheat detection Probe at 80C for 5min *#Add to sample and incubate at RT for 10min *#Wash two times for 1 minute each with 1 mL 1X PBS =====Padlock Probe capture, 2nd Rolony Process===== *#Strip Cy3-1st Rolony detection Probes with preheated 80% formamide at 70C, incubate for 15min *#Wash with PBS 3 times *#Prepare Agi26k0gap : 50nM in 120uL (Matt's product) --> 30uL in 100uL 2x SSC, which is 15nM *#*30uL Agi26k0gap (15nM) *#*10uL Ampligase (5 U/uL) *#*10uL Buffer *#*50uL H2O *#Add 100uL per MatTek dish, incubate dishes at 37C for 30min, and 45C for overnight *#Wash with PBS twice, *#Preheat 0.5uM FISSEQ_ppRCA primer in 2X SSC at 70C, and add and incubate it at 45C for 30min *#Wash with PBS twice *#Add RCA mix, incubate at 30C for overnight (start : 08/12 3pm) *#*H2O : 172uL *#*10x Buffer : 20uL *#*dNTP : 2uL *#*a-dUTP : 4uL *#*Phi29 (100 U/µl) : 2uL *#Wash with PBS once, add BS(PEG)9, incubate at room temp. for 1hr. *#Wash with PBS twice, add 1M Tris pH8.0 , incubate at room temp. for 1hr. *#Wash with PBS twice, store at PBS
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