Matt:LabNotes/2016-11-16

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DARTFISH on 5342 Occipital Cortex with SplintR in PA Gel[edit]

  • Try 1 sample with polyacrylamide gel added after permeabilization
  • Used 100nM padlock probe
  • Sample: GTEx Patient 5342 Occipital Cortex
Component Volume
40% AB 199:1 Mix 6.25
10% BSA 1
10mg/ml Acryloyl-X, SE in DMSO 1
H2O 39.75
5% TEMED 1
5% APS 1
Total 50

Protocol[edit]

Day 1[edit]

  1. Prepare plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH and UV sterilize
  2. Made 4% PFA in 1X PBS
    • 10ml 16% PFA + 4ml 10X PBS + 26ml H2O
  3. Take out brain section from -80C and dry on 50C hot plate for 3min
  4. Submerge coverslip in 5ml 4% PFA in petri dish for 15min at 37C
  5. Wash twice with cold 1X SSPE by submerging
  6. Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
  7. Wash with cold nf-H2O three times and check for degradation
  8. Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
    • 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
  9. Wash with nf-1X PBS three times and check for degradation
    • First try: ALL the tissue sloughed off by the end of 3rd wash
    • Second try: Decreased pepsin time to 60sec and only the areas circled in red in image below sloughed off so continued
      • Polymerization of gel was successful but when trying to remove coverslip from glass slide the coverslip cracked
    • Third try: With same pepsin time of 60sec all tissue sloughed off
    • Fourth try: Skipped pepsin step but then dropped coverslip face down on bench... SHIT!

File:IMG 0297.jpg

  1. Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue
    • Secure in plastic jig
  2. Add 50ul gel casting mix filtered and degassed
  3. Seal in plastic bag and vacuum out air before filling with argon
  4. Let sit at RT for 30min
  5. Aspirate non-polymerized gel and wash once with 1X PBS
  6. Attach coverslip to bottom of petri dish
  7. Aspirate non-polymerized gel and wash once with 1X PBS
  8. Prepare SplintR Mix
    • Preheat Padlock Probes then snap cool
Component Volume
10X SplintR Buffer 10
CA12kOct2016_V4 160nM 60
H2O 30
Total 100
  • Padlock probes are mixed with SplintR Buffer but since they weren't used put in 4C box labeled "Matt"
    • May need to spike in ATP next time it is used