Matt:LabNotes/2016-6-29

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Revision as of 23:49, 29 June 2016 by >Mzcai (Created page with "=FISSEQ on BA8 Sections with PA gel + Acrydite/BS(PEG)9= *Used worst section from 1/19/16 BA8 patient 1568 from Yun *Gel mix and protocol is same...")
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FISSEQ on BA8 Sections with PA gel + Acrydite/BS(PEG)9

not be below 6.5."

Protocol

Day 1

  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 cold 1X SSPE
  15. Attach coverslip to bottom of petri dish
  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