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=FISSEQ on Mouse Embryo Test= *Try 2 samples: slide5 (20um thickness) and slide6 (25um thickness) ==Embryo Section Info== #E7.5#3 slide1 is 16um (8 sections beginning of embryo + placenta) on the coverslip there is written A1, B1, C1, D1 to help for the orientation. #E7.5#3 slide2 is 20um (8 sections beginning of embryo + placenta) on the coverslip there is written A2, B2, C2, D2 to help for the orientation. #E7.5#3 slide3 is 25um (7 sections beginning of embryo + placenta) on the coverslip there is written A3, B3, C3, D3 to help for the orientation. #E7.5#3 slide4 is 16um (7 sections mainly embryo) on the coverslip there is written A4, B4, C4, D4 to help for the orientation. #E7.5#3 slide5 is 20um (7 sections middle embryo) on the coverslip there is written A5, B5, C5, D5 to help for the orientation. #E7.5#3 slide6 is 25um (7 sections middle embryo) on the coverslip there is written A6, B6, C6, D6 to help for the orientation. #E7.5#3 slide7 is 16um (10 sections middle embryo) on the coverslip there is written A7, B7, C7, D7 to help for the orientation. #E7.5#3 slide8 is 20um (12 sections middle/end embryo) on the coverslip there is written A8, B8, C8, D8 to help for the orientation. #E7.5#3 slide9 is 25um (6 sections end embryo+ adult liver) on the coverslip there is written A9, B9, C9, D9 to help for the orientation. ==Protocol== *For CircLigase reaction use [[Matt:JustinLabNotes/2015-10-7 | MnCl2 made by Justin]] and 5M Betaine from Sigma ===Day 1=== #Sterilize culture dishes and tweezers with EtOH, RNaseZap, and then rinse with nf-H2O #Attach 22mm x 22mm glass coverslips (with mounted sections) to bottom of culture dishes with 16mm diameter hole #*Use Double-sided tape: Adhesives Research IS-8458-19 (Hosuk originally mistakenly told me IS-7876-35) #Wash tissue section twice using cold nf-H2O for 5min each #Add 0.25% TX-100 in nf-H2O for 5min at RT #Wash with cold nf-H2O twice #Add 200ul 0.01% pepsin in 0.01N HCl (2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O) for 10min at RT #Wash with 2ml of cold nf-PBS three times #Prepare 2X Reverse Transcription Mix '''on ice''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H2O||157 |- | 10X M-MuLV Buffer||20 |- | 25mM dNTP||2 |- | 4mM aa-dUTP||2 |- | 100uM FISSEQ_RT||5 |- | RNase Inhibitor||2 |- | M-MuLV RTase||10 |- | Total||200 |} #Incubate 10min at 4C and then ~18hr at 37C #*Parafilm each dish and then put in plastic bag with wet tissue ===Day 2=== #Wash with 1X PBS twice #*Dishes had small leak so had to seal with gap-filling glue #Add 200ul BS(PEG)9 (20ul BS(PEG)9 stock + 980ul 1X PBS) and incubate 1hr at RT #Wash with 1X PBS twice #Quench with 1M Tris for 30min at RT #Wash with 1X PBS twice #RNA Removal #*Add 200ul RNase Mix (100ul RNase H 10X Buffer + 10ul Riboshredder + 50ul RNase H + 840ul H2O) and incubate 1hr at 37C #Wash with H2O twice #Add 200ul CircLigaseII mix and incuabate 3hr at 60C {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | DEPC-H2O||640 |- | CircLigase Buffer 10X||100 |- | MnCl2 50mM||50 |- | Betaine 5M|| 200 |- | CircLigase II (100U/ul)||10 |- | Total||1000 |} #Wash with 1X PBS twice #Add 200ul 0.5uM FISSEQ_RCA (5ul 100uM FISSEQ_RCA + 995ul 2X SSC + 30% formamide) and incubate 1hr at 60C #Wash with 2X SSC + 30% formamide for 10min at 60C #wash with 2X SSC, 1X SSC, 1X PBS once each #Add 200ul RCA mix and incubate for 15hr at 30C {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H2O||174 |- | 10X Phi29 Buffer||20 |- | 25mM dNTP||2 |- | 4mM aa-dUTP||2 |- | Phi 29 (low conc)||2 |- | Total||200 |} ===Day 3=== #Wash with 1X PBS once #Add 200ul BS(PEG)9 (20ul BS(PEG)9 stock + 980ul 1X PBS) and incubate 1hr at RT #Wash with 1X PBS twice #Quench with 1M Tris for 30min at RT #Wash with 1X PBS twice #Add 200ul 75C preheated 0.5uM FISSEQ_Adpt (Cy3) in 2X SSC + 30% formamide and incubate 10min at RT #Wash with 2X SSC twice #Image with Confocal ==Image Results== ===slide 5 20um=== *Section 1 10X [[File:Cropped_BF_20151009_E7.5_a5_20um_10X_Section1.jpg]] *Section 2 10X [[File:Cropped_BF_20151009_E7.5_a5_20um_10X_Section2.jpg]] *Section 1 20X [[File:MAX_20151009_E7.5_a5_20um_FISSEQ_20X_Cy3.jpg|450px]] [[File:Merge_MAXandBF_20151009_E7.5_a5_20um_FISSEQ_20X_Cy3.jpg|450px]] *Section 1 63X [[File:MAX_20151009_E7.5_a5_20um_FISSEQ_63X_Cy3.jpg|450px]] [[File:Merge_MAXandBF_20151009_E7.5_a5_20um_FISSEQ_63X_Cy3.jpg|450px]] *Section 2 had no fluorescence in Cy3 channel for no apparent reason ===slide 6 25um=== *10X [[File:Cropped_BF_20151009_E7.5_a6_25um_10X.jpg]] *63X [[File:MAX_20151009_E7.5_a6_25um_FISSEQ_63X_Pos2_Cy3.jpg|450px]] [[File:Merge_MAXandBF_20151009_E7.5_a6_25um_FISSEQ_63X_Pos2_Cy3.jpg|450px]]
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