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=Polyacrylamide Gel on Brain Tissue Sections with Argon Chamber Test= ==Purpose== *Use argon gas chamber (sealed plastic bag) to improve polymerization of polyacrylamide gel *Creating a low density polyacrylamide gel surrounding the tissue can keep tissue together while allowing diffusion of DNA oligos and enzymes *[[Matt:LabNotes/2016-2-10#Polyacrylamide_Gel_on_culture_dish | Previously]] tried minimizing oxygen exposure (which inhibits polymerization) by using vacuum chamber *[[Matt:LabNotes/2016-1-20 | Sample dish A & B]] ==Experiment== *Based on this general [[Matt:LabNotes/Polyacrylamide_Gel_Protocol]] #Turn on UV lamp in AirClean hood for 15 minutes. #Use "AB mix" from last week: ##180 uL IEF 40% Acrylamide (made from powder) ##20 uL Acrylamide / Bis (19:1; 38%:2%) #Using a 3-cc syringe and a 0.22 micron filter, filter ~200 uL of the AB mix into a 1.5 mL microcentrifuge tube. #Degas the AB mix by using vacuum and sonicator #Prepare fresh 5% APS (ammonium persulfate) ##5 mg APS β100 uL dH20 #Prepare fresh 5% TEMED (in fume hood) ##2 uL TEMED β 38 uL dH20. #Put a few drops of 10% BSA into a 1.5 mL tube (BSA should be stored at 4'C) #Prepare the gel-casting mix (200 uL total volume). Do not add APS until immedietely prior to casting the gels ##50 uL AB mix (FILTERED) ##4 uL 10% BSA ##138 uL dH20 ##4 uL 5% TEMED ##4 uL 5% APS #Aspirate PBS from sample and then add 150ul gel-casting mix #Put in plastic bag and seal #Use vacuum to suck out air and then add argon gas #Let sit in sealed bag for 30min #Repeated everything for Sample B but let 138ul spread out completely over coverslip ==Results/Observations== *Sample A: **It worked! >95% of gel mix polymerized **I could aspirate the soluble mix that remained on top and the gel will get sucked on but won't detach from glass **Since I didn't believe it would work so well I did not spread it out evenly across glass *Sample B: **It worked again! **There was actually a small droplet of polymerized gel from last time stuck to glass, picked it off **Some gel mix leaked under coverslip and polymerized, easily scraped away **Stuck nicely to glass when washing with 1X PBS ==Conclusion== *Using argon gas in plastic bag is needed for polymerization of thin gel on coverslip *Gel sticks to glass well *Still need to test if dye-probes can diffuse to tissue and be imaged clearly *Still need to test making thinner gel *Then need to test whether can do DARTFISH with gel right after or during fixation ==Imaged with Universal dye-probe 2-19-2016== *Hybridized 0.5uM FISGA_adpt (Cy3) in 2X SSC + 30% formamide *Imaged with 63X confocal
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