Matt:LabNotes/2016-2-18
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Polyacrylamide Gel on Brain Tissue Sections with Argon Chamber Test[edit]
Purpose[edit]
- 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
- Previously tried minimizing oxygen exposure (which inhibits polymerization) by using vacuum chamber
- Sample dish A & B
Experiment[edit]
- 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 →100 uL dH20
- Prepare fresh 5% TEMED (in fume hood)
- 2 uL TEMED → 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[edit]
- 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[edit]
- 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[edit]
- Hybridized 0.5uM FISGA_adpt (Cy3) in 2X SSC + 30% formamide
- Imaged with 63X confocal