Matt:LabNotes/2015-8-4

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Revision as of 02:13, 5 August 2015 by >Mzcai (Created page with "=BA8 Section DARTFISH with Fiducial Beads= *Try again: Matt:LabNotes/2015-8-3 **with fresh PFA *Practice DARTFISH on BA8 tissue sections *Use FluoSpheres Amine-Modified M...")
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BA8 Section DARTFISH with Fiducial Beads

  • Practice DARTFISH on BA8 tissue sections
  • Use FluoSpheres Amine-Modified Microspheres 0.2um (505/515) as fiducial markers

DARTFISH on BA8

  1. Prepare plastic culture dish with hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH sterilize and UV
  2. Made 40ml 4% PFA
    • 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O
  3. Take out BA8 section from -80C and incubate on 50C hot plate for 3min
  4. Submerge section in 5ml 4% PFA in 6 well culture plate for 15min at 37C
  5. Wash with 2X SSPE by submerging in 2 clean wells of culture plate with 5ml SSPE
  6. Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
    • Still had leak so used gap-filling adhesive 200 to seal
  7. Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
    • 0.25% TX-100 in 2XSSPE tube had crack in cap so not completely sealed
  8. Wash with nuclease-free H2O three times
  9. Add 0.01% Pepsin in 0.1N HCl and incubate for 5min at 37C
    • 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
  10. Wash with nuclease-free PBS three times
    • Can already see tissue is degrading
  11. Prepare Reverse Transcription Mix on ice
Components Volume
H2O 157
10X M-MuLV Buffer 20
25mM dNTP 2
2mM aa-dUTP 4
100uM N9 5
RNase Inhibitor 2
M-MuLV RTase 10
Total 200
  1. Incubate 10min at 4C and then ~15hr at 37C
  • Tissue is degrading too easily and PFA shouldn't be the problem
    • Maybe 0.25% TX-100 has higher concentration than labeled because of evaporation, will try making new one
    • Or skip pepsin digestion?
    • Could it be due to long storage time?