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=FISSEQ on BA8 sections with PA gel + Acrydite/BS(PEG)9= *Try FISSEQ to see a "baseline" of number of rolonies *Gel mix and protocol is same as [[Matt:LabNotes/2016-6-28#Gel_Mix|this with BS(PEG)9]] {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:11pt" valign="bottom" | width="51" height="14" | Component | width="51" | Volume |- style="font-size:11pt" valign="bottom" | height="14" | 40% AB 199:1 Mix | align="right" | 6.25 |- style="font-size:11pt" valign="bottom" | height="14" | 10% BSA | align="right" | 1 |- style="font-size:11pt" valign="bottom" | height="14" | 1mM Acryd/Amine Linker | align="right" | 6.25 |- style="font-size:11pt" valign="bottom" | height="14" | H2O | align="right" | 33.5 |- style="font-size:11pt" valign="bottom" | height="14" | 5% TEMED | align="right" | 1 |- style="font-size:11pt" valign="bottom" | height="14" | 5% APS | align="right" | 1 |- style="font-size:11pt" valign="bottom" | height="14" | BS(PEG)9 | align="right" | 1 |- style="font-size:11pt" valign="bottom" | height="14" | Total | align="right" | 50 |} ==Protocol== ===Day 1=== #Prepare a plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate #*EtOH and UV sterilize #Use 4% PFA in 1X PBS from 7/6/2016 #Take out BA8 section from -80C and dry on 50C hot plate for 3min #Submerge coverslip in 5ml 4% PFA in 6 well culture plate for 15min at 37C #Wash twice with cold 1X SSPE by submerging #Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT #Wash with cold nf-H2O three times '''and check for degradation''' #Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C #*2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O #Wash with nf-1X PBS three times '''and check for degradation''' #Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue #*Secure in plastic jig #Add 50ul gel casting mix '''filtered and degassed''' #*5% gel with 250uM Acrydite-Amine linker and 5mM BS(PEG)9 #Seal in plastic bag and vacuum out air before filling with argon #Let sit at RT for 30min #Aspirate non-polymerized gel and wash once with 1X PBS #*[[Photo here]] #Attach coverslip to bottom of petri dish #Aspirate non-polymerized gel and wash once with 1M Tris and once with cold 1X SSPE #Prepare Reverse Transcription Mix '''on ice''' and add {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H2O||159 |- | 10X M-MuLV Buffer||20 |- | 25mM dNTP||2 |- | 4mM aa-dUTP||2 |- | 100uM FISSEQ_RT primer||5 |- | RNase Inhibitor||2 |- | M-MuLV RTase||10 |- | Total||200 |} #Incubate 10min at 4C and then ~18hr at 37C ===Day 2=== *[[File:20160714_BA8_Hydrogel_Day2.JPG|250px]] **No swelling **Good coverage of gel, almost no air bubbles, only edges lack gel which is not a problem #Wash with 1X PBS once #Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT #Wash with 1X PBS twice #Add 1M Tris pH 8.0 and incubate 30min at RT #Wash with 1X PBS twice #RNA Removal #*Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C #Wash with nf-H2O twice #Prepare Circligase mix on ice {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | DEPC-H2O||128 |- | CircLigase II Buffer 10X||20 |- | MnCl2 50mM||10 |- | Betaine 5M||40 |- | CircLigase II 100U/ul||2 |- | Total||200 |} #Add mix to sample and incubate for 3hr at 60C #Wash with 1X PBS twice #Add 200ul (1ul 200uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C #Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS #Prepare RCA reaction mix on ice {| {{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 |} #Add RCA mix and incubate at 30C overnight (~15hrs) ===Day 3=== #Wash with 1X PBS once #Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr #Wash with 1X PBS twice #Add 1M Tris pH 8.0 and incubate at RT for 30min #Wash with 1X PBS twice ===Imaging=== #Add 0.5uM FISSEQ_Adpt in 30% formamide + 2X SSC preheated to 75C #Incubate for 10min at RT #Wash with 2X SSC twice #Image (saved in 7-20-2016) ==Results== *Took images at 6 positions **Tissue approximately outlined in yellow [[File:20160720_BA8_FISSEQ_50umGel_Positions.jpg|450px]] ===Position 1=== [[File:MAX_20160720_BA8_FISSEQ_50umGel_Pos1.jpg|350px]][[File:20160720_BA8_FISSEQ_50umGel_Pos1_z00_ch01.jpg|350px]] ===Position 2=== [[File:MAX_20160720_BA8_FISSEQ_50umGel_Pos2.jpg|350px]][[File:20160720_BA8_FISSEQ_50umGel_Pos2_z06_ch01.jpg|350px]] ===Position 3=== [[File:MAX_20160720_BA8_FISSEQ_50umGel_Pos3.jpg|350px]][[File:20160720_BA8_FISSEQ_50umGel_Pos3_z05_ch01.jpg|350px]] ===Position 4=== [[File:MAX_20160720_BA8_FISSEQ_50umGel_Pos4.jpg|350px]][[File:20160720_BA8_FISSEQ_50umGel_Pos4_z03_ch01.jpg|350px]] ===Position 5=== [[File:MAX_20160720_BA8_FISSEQ_50umGel_Pos5.jpg|350px]][[File:20160720_BA8_FISSEQ_50umGel_Pos5_z05_ch01.jpg|350px]] ===Position 6=== [[File:MAX_20160720_BA8_FISSEQ_50umGel_Pos6.jpg|350px]][[File:20160720_BA8_FISSEQ_50umGel_Pos6_z04_ch01.jpg|350px]] ==Conclusion== *Dropping polyacrylamide mix on coverslip before sandwiching with glass slide works well to prevent bubbles *Less rolonies than previous FISSEQ on BA8 tissue sections
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