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=DARTFISH on BA8 with and without PA Gel= *Try 3 samples with polyacrylamide gel added at different points in protocol *Sample 1: No gel *Sample 2: Gel added after permeabilization *Sample 3: Gel added after RNA removal *Used 200nM padlock probe (twice the normal concentration) *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 3 plastic culture dishes with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate #*EtOH and UV sterilize #Make 4% PFA in 1X PBS #*10ml 16% PFA + 4ml 10X PBS + 26ml H2O #Take out BA8 section from -80C and dry on 50C hot plate for 3min #Submerge coverslip in 5ml 4% PFA in petri dish 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''' ====To Sample 2 Only==== *Skip these steps and go to Reverse Transcription Immediately for other 2 samples #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 125uM 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 #Attach coverslip to bottom of petri dish #Aspirate non-polymerized gel and wash once with 1M Tris and once with cold 1X PBS ====Continue for All Samples==== #Attach coverslip to bottom of petri dish #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 N9 primer||5 |- | RNase Inhibitor||2 |- | M-MuLV RTase||10 |- | Total||200 |} #Incubate 10min at 4C and then ~15hr at 37C ===Day 2=== #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 ====To Sample 3 Only==== #Add 50ul gel casting mix '''filtered and degassed''' #*5% gel with 125uM Acrydite-Amine linker, 5mM BS(PEG)9, and Use [[Matt:LabNotes/2016-7-26#Qubit_ssDNA | 478nM TB12kApr2016 V4 Probes]] instead of H2O (33.5ul) #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 1M Tris #Prepare Ampligase mix on ice #*Preheat Padlock Probes then snap cool before adding Ampligase buffer and enzyme {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Ampligase Buffer||10 |- | [[Matt:LabNotes/2016-7-26#Qubit_ssDNA | 478nM]]||80 |- | Ampligase||10 |- | Total||100 |} #Add mix to sample and incubate for 30min at 37C #Move sample to 60C and oven slowly decreases to 55C and held for ~24hr ====To Samples 1 & 2==== #Prepare Ampligase mix on ice #*Preheat Padlock Probes then snap cool before adding Ampligase buffer and enzyme {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Ampligase Buffer||10 |- | [[Matt:LabNotes/2016-7-26#Qubit_ssDNA | 478nM]]||80 |- | Ampligase||10 |- | Total||100 |} #Add mix to sample and incubate for 30min at 37C #Move sample to 60C and oven slowly decreases to 55C and held for ~44hr ===Day 3=== #Wash with 1X PBS twice #Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) pre-heated to 65C and incubate 1hr at 55C #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 4=== #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 8-8-2016) ====Notes==== *Laser intensity = 1%, Cy3 gain = 600 (tried increasing gain but only increased background) *Z-stack system optimized for 63X objective (0.30um step size) *Imaged Samples 1, 2, and 3 in that order *Imaged each from bottom of white matter to top-left of white matter *Sample 1 also imaged up-left of white matter to show degradation and lack of rolonies in grey matter *Sample 1 had thickness <10um, thicker in white matter obviously *Sample 2 shows odd round objects in BF image when in white matter area **For some reason these are not seen in areas that are definite grey matter but I am not sure where the white/grey matter boundary is *Sample 3 had thickest z-stack probably because gel was made thicker and rolonies were generated in gel but above tissue ====Bottom==== ====Bottom-Mid==== ====Mid==== ====Top Left==== ====Grey Matter==== ===Decoding Sample 2: Imaging=== ====Add [[Matt:LabNotes/2015-8-13#Add_Beads| 1:2000 diluted Fiducial Beads]]==== #Strip with pre-heated 80% formamide twice for 15 min each #Wash with 1X PBS twice #Wash with 10mM HEPES #Get aliquoted 1:2000 diluted beads from dcProbe box in 4C #Vortex (normally should sonicate) #Add to sample and incubate 5min at RT #Wash with 10mM HEPES once #Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 20mM HEPES) and incubate at RT for 1hr #Wash with 1X PBS twice #Add 1M Tris pH 8.0 and incubate at RT for 30min *Did not detect any beads, may need to allow longer incubation due to gel ====Notes==== *22 images 170um away from each other **184umx184um FOV so ~14um (<10%) overlap between images *By the time I finish defining z-start and z-end positions the stage has drifted **Seems like stage drifts down ~3um per hour **Need to define >3um below what I want and leave extra room above and below *Left sample overnight and it dried out (gel was all cracked) **Rehydrated in 2X SSC for 1hr ***Tissue morphology looks the same ***Fewer/less rolony signal but this could be due to 2 other factors *20160810_S2_BA8_DARTFISH_TB12kApr2016V4_Cycle# **Cycle0 = Universal rolony probe (Cy3) **Cycle1-6 for decoding **Cycle7 for DRAQ5 *Pos1 of 22 with approx position of tissue section outlined in red [[File:20160810_22pos_DARTFISH_BA8_Pos1.JPG.JPG|350px]] *Pos22 of 22 with approx position of imaged tile outlined in red [[File:20160810_22pos_DARTFISH_BA8_Pos22.JPG|350px]] =====Cycle2===== *Retook Position19019 and Position20020 because z range was offset, started from middle of signal =====Cycle6===== *Retook Position20020 and Position21021 because z range was offset, started from middle of signal ===Decoding Sample 2: Image Analysis=== *331 GB total size of raw images *Due to time constraints during imaging, could not export raw tiff files from Leica software during imaging process **Each cycle would need >30min to export and I already use all the downtime to save the experiment in Leica format #So first thing to do was reopen each experiment in Leica and then export as tiff #*Each cycle saved as its own experiment because Leica software has limited memory #*Each cycle takes about 2 hours to open and then export #Then uploaded raw data from local computer to ConfocalStore server #Then downloaded from ConfocalStore server to my laptop's external hard drive #Replaced Cycle2 Pos19 & Pos20 and Cycle6 Pos20 & Pos21 with retaken images #*i.e. replace 20160810_S2_BA8_DARTFISH_TB12kApr2016V4_Cycle2\Position19019\ with 20160810_S2_BA8_DARTFISH_TB12kApr2016V4_Cycle2_Pos19-20\Position19019\ #First try 2D decoding because much faster #Make Max Intensity Z Projection for every stack #*[[Media:BatchMIP.txt|BatchMIP.py]] <!-- ==Results== -->
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