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===Rolony from Mouse Brain Tissue Section : 9th Try=== *8th test was failed necause of mistake (not adding RT primers) *Tisses that I've got were fixed with 4% Formaldehyde (by Yun) *High Viscosity!!!) of Cyanoacrylate adhesive (Sigma-adrich, P/N:Z105902-1EA) is perfect !!! ====Procedure==== *06/25 *#Put DEPC-PBS in 3 wells of 6-well plate, and wash fixed tissue y deeping in series of PBS *#Dry PBS from cover slip and attach the glass cover slip with a mounted tissue section to the MatTek dish using cyanoacrylate adhesive (High Viscosity!!!) *#Wash twice using DEPC-treated H2O for 5 min each *#Add 0.25% Triton X-100 in DEPC-treated H2O for 15 min (S1, 10min for S2) and aspirate *#Wash with DEPC-treated H2O twice *#Add 200 ul 0.1% pepsin in 0.1 N HCl for '''15 min''' at 37C *#Wash with 2 ml 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 4Β°C, then transfer to 37Β°C for 12 hr. *06/26 *#Wash with PBS once, skip BS(PEG)9 *#Add RNase H mix, incubate for 1hr. at 37C *#*RNase H mix : 2 ul Riboshredder (Epicentre)+ 10 ul RNase H(Enzymatics) in 20 ul RNase H buffer(Enzymatics) + 168 uL H2O *#Wash with nucleaus-free H2O twice *#Add CircLigase II mix, incubate at 60C for 2hr. *#*H2O 148uL *#*Buffer 20uL *#*50mM MnCl2 10uL *#*5M Betaine 20uL (0.5M in final reaction volume, according to Nat. Protocol paper by Jay) *#*CircLigaseII 2uL *#RCA Primer, final concentration : 0.5uM in 200uL (2x SSC + 30% Formamide) at 60C for 1hr. --> 1uL of 100uM RCA Primer + 199uL 2X SSC with 30% Formamide *#1uL of 100uM RCA Primer + 199uL 2X SSC with 30% Formamide *#Aspirate and add 2XSSC, incubate at 60C for 10min *#Aspirate and Wash with 2X SSC, 1X SSC and PBS once each *#Prepare RCA reaction mix *#*H2O 172uL *#*Buffer 20uL *#*25mM dNTP 2uL *#*2mM a-dUTP 4uL *#*Phi29 2uL *#Add RCA mix, incubate at 30C for 18hr *06/27 *#Wash with PBS once, Prepare 4uL of BS(PEG)9 in 196uL PBS, add 200uL BS(PEG)9 mix, incubate for 1hr at RT *#Wash with PBS once, add Tris pH 8.0, incubate for 30min *#Wash with PBS once *#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 *#Imaging ====Result==== *Both samples, 10um and 20um thickness, stayed well. *But the rolonies were not many. 20um thickness has less rolonies, weaker signal than 10um does. **Probably permeablization was not enough! [[File:RolonyCountTable_2014-06-27.jpg|200px]] [[File:Tissue_9th_T10um_20um_2014-06-27.jpg|700px]] ====Issue of tile imaging by confocal==== *A lot of artifact along boundaries, it might be uneven brigtness. *Tile image (Bright field) by PMT-Trans, 10x obj. 512x512 pixel *Sample1 [[File:Confocal_Tile_10xobj_Sample1_06272014.jpg|600px]] *Sample2 [[File:Confocal_Tile_10xobj_Sample2_06272014.jpg|600px]] * Tile image of Sample 2 (Bright field) by Epi-microcope, 4x obj. (Manually moved) [[File:Epi_4xobj_Sample2_BF_2014-06-27.jpg|700px]]
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