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=DARTFISH on BA8 Sections with PA gel= *[[Matt:LabNotes/2016-1-20|Last time concluded I need to cover tissue in thin gel]] *Used worst section from [[Matt:LabNotes/2016-1-20|1/19/16 BA8 patient 1568 from Yun]] *Gel mix and protocol is same as [[Matt:LabNotes/2016-2-18|this successful experiment]] *4% Formaldehyde is one month old and has been sitting at RT **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 1/20/2016 #Take out second worst BA8 sections (second furthest front) 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 100ul gel casting mix '''filtered and degassed''' #Cover with plastic coverslips cut and glued together such that it leaves ~0.3mm 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 #*Mostly did not polymerize #*Only a ~1cm diameter circle of gel polymerized in center #*I will continue with FISSEQ protocol to see whether can generate as many rolonies in gel area as non-gel area #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''' #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=== *Gel must have expanded overnight and created a "tent" in the center of glass where gel is pushed up **The swelling is only on the grey matter (superficial layers) of tissue and nowhere else (white matter and glass) but could just be coincidence? #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 *B13: 305nM **This batch of padlock probes was PCR'd an extra 3 cycles during production PCR {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | DEPC-H2O||18.43 |- | Ampligase Buffer||10 |- | Erin's Batch 13 2/12/2016 Padlock Probes 305nM]]||33 |- | 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 ~20hr ===Day 3=== #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 18hr 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 3-4-2016) #*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 2mm from edge #*Bottom of P wasn't covered by gel so image that as well #Strip with 80% formamide preheated to 75C #Wash with 1X PBS twice ====0mm from edge==== [[File:MAX_20160229_BA8_DARTFISH_0mm_edge.jpg|450px]][[File:20160229_BA8_DARTFISH_0mm_edge_z24_ch01.jpg|450px]] ====2mm from edge==== [[File:MAX_20160229_BA8_DARTFISH_2mm_edge.jpg|450px]][[File:20160229_BA8_DARTFISH_2mm_edge_z24_ch01.jpg|450px]] ====4mm from edge==== [[File:MAX_20160229_BA8_DARTFISH_4mm_edge.jpg|450px]][[File:20160229_BA8_DARTFISH_4mm_edge_z21_ch01.jpg|450px]] ====6mm from edge==== [[File:MAX_20160229_BA8_DARTFISH_6mm_edge.jpg|450px]][[File:20160229_BA8_DARTFISH_6mm_edge_z22_ch01.jpg|450px]] ====8mm from edge==== [[File:MAX_20160229_BA8_DARTFISH_8mm_edge.jpg|450px]][[File:20160229_BA8_DARTFISH_8mm_edge_z17_ch01.jpg|450px]] ====bottom==== [[File:MAX_20160229_BA8_DARTFISH_bottom_nogel.jpg|450px]][[File:20160229_BA8_DARTFISH_bottom_nogel_z21_ch01.jpg|450px]] ===Conclusion=== *Compare with [[Matt:LabNotes/2016-1-20#Imaging_Results]] *Tissue definitely had less degradation due to gel *However also much fewer rolonies than with gel *It is possible that gel prevented proper permeabilization and pepsin digestion since I did not change those times **Pepsin digestion time especially may need to be increased *Solution: Fix and pepsin digest BEFORE adding PA gel
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