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=DARTFISH on BA8 Sections= *Try one sample with second fixation step after permeabilization with TX-100 and Pepsin *If sample is good use to compare with Gwen's RNAscope images of BA8 with 4 genes: OLFM1, PDE1A, SCL17A7, RELN ==Protocol== ===Day 1=== #Prepare 2 plastic culture dishes with 17mm hole, tweezers, large plastic dish, and 50C hot plate #*EtOH and RNase Free sterilize, then rinse with copious amounts of MilliQ water #Made 40ml 4% PFA at RT #*10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O #Take out BA8 sections (2 coverslips) from -80C and incubate on 50C hot plate for 3min #Submerge coverslips in 5ml 4% PFA in 6 well culture plate for 15min at 37C #Wash twice with cold 1X PBS by submerging #Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive #*One glass cracked so only one sample left '''DO SECOND FIXATION''' #Add cold 0.25% TX-100 in 2XSSPE and incubate 10min at RT #Wash with cold nf-H2O three times #Add 0.01% Pepsin in 0.1N HCl and incubate for 5min at 37C #*2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O #Wash with cold 1X PBS three times #Add 500ul 4% PFA for 10min at RT and #Wash with cold 1X PBS three times #Prepare 2X 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 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | DEPC-H2O||20 |- | Ampligase Buffer||10 |- | Justin's Batch 5 7/22/2015 Padlock Probes 320nM]]||26.3 |- | Justin's Batch 7 8/31/2015 Padlock Probes 329nM]]||5 |- | 100nM suppv2 Oligos || 28.57 |- | Ampligase||10 |- | Total||100 |} #Add mix to sample and incubate ~1hr at 60C as oven slowly decreases to 55C and held for another ~20hr ===Day 3=== #Wash with 1X PBS twice #To DARTFISH slide 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 10.5hr 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 |} #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 and store in 1X PBS at 4C ===Day 4=== #Add 200ul 0.5uM FISGA_Adpt in 2X SSC + 30% Formamide pre-heated to 75C and incubate for 10min at RT #Wash with 2X SSC twice #Add 400ul 2X SSC for imaging ===10-26-2015=== *When taking look at dish, noticed it was starting to leak and tried to seal again with superglue *In the process, cracked the glass and had to throw away ==Results== *Second fixation definitely helped retain tissue integrity **Noticed much less tissue being washed away *However number of rolonies is very few per cell ===Images=== *Tissue was oriented so the long axis was parallel with the y axis and the wider end was pointed "north" and the skinny end was "south" *Right edge (East) is very straight and from deeper layer of cortex *Left edge (West) is curved and rougher and from superficial layer of cortex *Pos 1 is in the middle near the right edge where I saw the most tissue left on glass (opaque white) *Pos 2-5 goes from deep layer to superficial layer at skinny end of tissue "south" ====Pos 1==== *Random position near center of tissue [[File:MAX_20151015_BA8_DARTFISH_Pos1_Gain600.jpg.jpg|450px]] [[File:MAX_cropped_20151015_BA8_DARTFISH_Pos1_Gain600.jpg]] ====Pos 2==== *Near deep side (layer 6?) of skinnier end of section [[File:MAX_20151015_BA8_DARTFISH_Pos2_Gain550.jpg|450px]] ====Pos 3==== [[File:MAX_20151015_BA8_DARTFISH_Pos3_Gain550.jpg|450px]] ====Pos 4==== [[File:MAX_20151015_BA8_DARTFISH_Pos4_Gain550.jpg|450px]] ====Pos 5==== *Near superficial side (layer 1?) of skinnier end of section [[File:MAX_20151015_BA8_DARTFISH_Pos5_Gain550.jpg|450px]]
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