Matt:LabNotes/2016-11-29

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Revision as of 00:55, 7 December 2016 by >Mzcai (→‎Imaging)
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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 (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[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. 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

Day 2[edit]

  1. Wash with 1X PBS twice preheated to 55C
  2. Add SplintR Enzyme and incubate at 37C for 20min
    • 20ul 10X Buffer + 6ul SplintR + 174ul H2O
  3. Wash with 1X PBS twice
  4. Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) pre-heated to 65C and incubate 1hr at 55C
  5. Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
  6. Prepare RCA reaction mix on ice
Component Volume
H2O 174
10X Phi29 Buffer 20
25mM dNTP 2
4mM aa-dUTP 2
Phi 29 (low conc) 2
Total 200
  1. Add RCA mix and incubate at 30C overnight (~15hrs)

Day 3[edit]

  1. Wash with 1X PBS once
  2. Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr
  3. Wash with 1X PBS twice
  4. Add 1M Tris pH 8.0 and incubate at RT for 30min
  5. Wash with 1X PBS twice
  6. Sonicate 1:4000 diluted fluorescent spheres in bath sonicator
  7. Wash sample with 10mM HEPES
  8. Add beads to sample and incubate 5min at RT
  9. Wash sample with 10mM HEPES twice
  10. Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr
  11. Wash with 1X PBS twice
  12. Add 1X Tris pH 8.0 for 30min at RT
  13. Wash with 1X PBS twice

Imaging[edit]

  1. Add 0.5uM FISGA_Adpt in 30% formamide + 2X SSC preheated to 75C
  2. Incubate for 10min at RT
  3. Wash with 2X SSC twice

Imaged on 12-5-2016

Results[edit]

Conclusion[edit]