Matt:LabNotes/2016-3-28

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DARTFISH on BA8 Sections with PA gel[edit]

  • This time do tissue permeabilization before gel polymerization
  • Don't use 4% PFA in the hydrogel solution
    • I don't think swelling will be too big an issue

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 3/8/2016
  3. Take out second best remaining BA8 sections (5th from back) 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. Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
  7. Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
  8. Wash with cold nf-H2O three times and check for degradation
  9. Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
    • 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
  10. Wash with nf-1X PBS three times and check for degradation
  11. Add 75ul gel casting mix filtered and degassed
    • 10% Acrylamide/Bis-Acrylamide (199:1), 0.2% BSA, 0.1% APS, 0.1% TEMED
  12. Cover with plastic coverslips cut and glued together such that it leaves ~0.25mm space for gel height
  13. Seal in plastic bag and vacuum out air before filling with argon
  14. Let sit at RT for 30min
  15. Aspirate non-polymerized gel and wash once with cold 1X SSPE
    • Edges did not polymerize but the grey matter is fully covered
  16. Prepare Reverse Transcription Mix on ice and add
Components Volume
H2O 159
10X M-MuLV Buffer 20
25mM dNTP 2
4mM aa-dUTP 2
100uM N9 5
RNase Inhibitor 2
M-MuLV RTase 10
Total 200
  1. Incubate 10min at 4C and then ~18hr at 37C

Day 2[edit]

  • There was almost no solution left on coverslip though there was around edges of dish
    • Was it bumped, tilted, or moved by someone?
  • Gel swollen and wrinkled as expected
  1. Wash with 1X PBS once
  2. Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT
  3. Wash with 1X PBS twice
  4. Add 1M Tris pH 8.0 and incubate 30min at RT
  5. Wash with 1X PBS twice