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=DARTFISH on BA8 with SplintR in PA Gel= *Try 1 sample with polyacrylamide gel added [[Matt:LabNotes/2016-8-3|after permeabilization]] *Used 200nM padlock probe (twice the normal concentration) *5% Gel mix and protocol is same as [[Matt:LabNotes/2016-6-28#Gel_Mix|this with Acryloyl-X]] {| 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" | 10mg/ml Acryloyl-X, SE in DMSO | align="right" | 1 |- style="font-size:11pt" valign="bottom" | height="14" | H2O | align="right" | 39.75 |- 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" | Total | align="right" | 50 |} ==Protocol== ===Day 1=== #Prepare plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate #*EtOH and UV sterilize #Made 4% PFA in 1X PBS on 8/31/2016 #*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''' #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''' #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 1X PBS #Prepare SplintR Mix #*Preheat Padlock Probes then snap cool {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X SplintR Buffer||10 |- | [[Matt:LabNotes/2016-9-14#EtOH_Precipitation_2 | 268nM]]||80 |- | H2O||10 |- | Total||100 |} #Move sample to 60C and oven slowly decreases to 55C and held for ~24hr ===Day 2=== #Wash with 1X PBS twice preheated to 55C #*Sample was dry in center, unusual amount of condensation on lid. Image borders of tissue should be okay. #Add SplintR Enzyme and incubate at 37C for 20min #*20ul 10X Buffer + 6ul SplintR + 174ul H2O #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 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 FISGA_Adpt in 30% formamide + 2X SSC preheated to 75C #Incubate for 10min at RT #Wash with 2X SSC twice #Image (saved in 9-23-2016) #*Taken with zstep = 1um except 'Edge' with zstep = 0.3um #3 Images taken #*Middle - the area that dried up during padlock probe capture #*Edge - Close to superficial layer that most likely did not dry #*Between - Somewhere in between middle and edge ==Results== ===Middle=== [[File:MAX_Middle.jpg|650px]] ===Between=== [[File:MAX_Between.jpg|650px]] ===Edge=== [[File:MAX_Edge.jpg|650px]] ==Conclusion== *Rolony density is higher than ever seen before with FISSEQ or DARTFISH **Especially in 'edge' image, which is near superficial layer 1 of neocortex that normally has least amount of rolonies *Specificity probably good because of nuclear enrichment of rolonies **If non-specific then wouldn't see so much more in nucleus than anywhere else *Decreased rolony density in middle is most likely due to dried up during padlock probe annealing and not indicative of poor results *NEXT STEP: Decode this sample to prove real rolonies and then Order New Padlock Probes that is RevComp of TB12k_Apr2016
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