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=DARTFISH on BA8 Sections with PA gel= *[[Matt:LabNotes/2016-2-29|Last time using gel to cover section it swelled during RT incubation]] **To prevent swelling going to use 4% formaldehyde in the gel solution, which crosslinks the hydrogel to Vectabond treated glass (amine functional groups) **Also add hydrogel '''after''' permeabilization because I think last time the hydrogel prevented effective permeabilization *Used 4th best section from [[Matt:LabNotes/2016-1-20|1/19/16 BA8 patient 1568 from Yun]] *4% Formaldehyde for fixation is one week old and has been sitting at RT under argon gas **From <http://swehsc.pharmacy.arizona.edu/sites/swehsc.pharmacy.arizona.edu/files/docs/ci/formaldehyde.pdf> **"We recommend that 10% buffered formalin solutions be used no longer than 3 months after they were initially mixed. The solution should be clear, colorless, with no precipitate and the pH should not be below 6.5." ==Protocol== ===Day 1=== #Prepare plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate #*EtOH and UV sterilize #Used 4% PFA from 3/8/2016 #Take out second best remaining BA8 sections (4th from back) 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 #Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive #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''' #Add 75ul gel casting mix '''filtered and degassed''' #*4% PFA, 10% Acrylamide/Bis-Acrylamide (199:1), 0.2% BSA, 0.1% APS, 0.1% TEMED #Cover with plastic coverslips cut and glued together such that it leaves ~0.25mm space for gel height #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 cold 1X SSPE #*Edges did not polymerize but the grey matter is fully covered #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||5 |- | RNase Inhibitor||2 |- | M-MuLV RTase||10 |- | Total||200 |} #Incubate 10min at 4C and then ~18hr 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 #Prepare Ampligase mix on ice #*Preheat H2O + Padlock probes + suppv2 Oligos to 85C and then snap cool before adding Ampligase buffer and enzyme *B9: 216nM {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | DEPC-H2O||5.1 |- | Ampligase Buffer||10 |- | Justin's Batch 9 9/11/2015 Padlock Probes 216nM]]||46.3 |- | 100nM suppv2 Oligos || 28.57 |- | 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 ===Day 3=== *Center of gel was not covered with liquid when taking out #Wash with 1X PBS once #Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C #*Preheated to 70C before adding #Wash with 2X SSC once, 1X SSC once, and 1X PBS once #Add RCA mix and incubate 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 4=== #Wash with 1X PBS once #Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 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 ===Check Rolony=== #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 3-18-2016) #*As usual have the tissue oriented like a backwards 'P' #*Image on horizontal line at widest section of 'P' #**Edge is right side of backwards 'P' (ie the vertical edge) #**Take images every certain distance from edge #*Bottom of P wasn't covered by gel so image that as well ===Results=== *Images not shown because no rolonies anywhere *Only some autofluorescence or non-specific fluorescence ===Conclusion=== *My guess is that the 4% PFA in hydrogel solution over cross-linked RNA during gel polymerization? *This prevents reverse transcription
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