Matt:LabNotes/2016-11-29

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DARTFISH on 5342 Occipital Cortex with SplintR in PA Gel

  • Try 1 sample with polyacrylamide gel added after permeabilization
  • Used 100nM padlock probe (mix made previously)
  • Sample: GTEx Patient 5342 Occipital Cortex sectioned by Yun on 11-22-2016
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

Day 1

  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. Skip pepsin step usually done here
  9. Aspirate any liquid and place a glass slide with 50um spacer over the coverslip with tissue
    • Secure in plastic jig
  10. Add 50ul gel casting mix filtered and degassed
  11. Seal in plastic bag and vacuum out air before filling with argon
  12. Let sit at RT for 30min
  13. Aspirate non-polymerized gel and wash once with 1X PBS
  14. Attach coverslip to bottom of petri dish
  15. Aspirate non-polymerized gel and wash once with 1X PBS
  16. Prepared SplintR Mix previously
    • Preheat Padlock Probes then snap cool
Component Volume
10X SplintR Buffer 10
CA12kOct2016_V4 160nM 60
H2O 30
Total 100
  1. Move sample to 60C HybEZ oven and slowly decreases to 55C and held for ~24hr