Matt:LabNotes/2016-6-29

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FISSEQ on BA8 Sections with PA gel + Acrydite/BS(PEG)9[edit]

not be below 6.5."

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. Used 4% PFA from 6/15/2016
  3. Take out worst BA8 section that is left from -80C and dry on 50C hot plate for 3min
  4. Submerge coverslip in 5ml 4% PFA in 6 well culture plate 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
  10. Aspirate any liquid and place a glass slide with 120um spacer over the coverslip with tissue
    • Secure in plastic jig
  11. Add 100ul gel casting mix filtered and degassed
    • 5% gel with 500uM Acrydite-Amine linker and 5mM BS(PEG)9
  12. Seal in plastic bag and vacuum out air before filling with argon
  13. Let sit at RT for 30min
  14. Aspirate non-polymerized gel and wash once with 1X PBS
  15. Attach coverslip to bottom of petri dish
    • COVERSLIP CRACKED DIAGONALLY WHEN CLEANING AWAY GEL
    • NEED TO BE MORE CAREFUL WHEN HANDLING
  • Continued by testing for swelling
  1. Put each half of cracked coverslip in petri dish with 1M Tris and put in 37C overnight

Results[edit]

File:20160630 GelTissueSwellTest 1.JPG File:20160630 GelTissueSwellTest 2.JPG

  • Next morning the gels were still stuck to glass
  • No significant swelling
    • Vertical length of gel was ~17mm when cast (no image but that is the length of the mold) and is ~17mm after overnight incubation in Tris
    • Suggests that the gel is covalently bound to glass

Conclusion[edit]

  • Making gel with Acrydite/Amine linker and BS(PEG)9 is viable solution to covalently bond polyacrylamide gel to Vectabond treated glass and tissue sections