Matt:LabNotes/2016-5-17

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Revision as of 23:11, 17 May 2016 by >Mzcai (→‎Day 1)
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FISSEQ whole mount embryo

  • Objective:
    • Test multiple permeabilization conditions
    • Embed in thin PA hydrogel for imaging (FISH and stripping)

Procedure

  • Embryos are fresh and sitting in 1X PBS in 200ul PCR tube

Day 1

  1. Sterilize bench and tweezers with EtOH, RNaseZap
  2. Make fresh 4% PFA in PBS
    • 10ml 16% PFA + 4ml 10X PBS + 26ml nfH2O
    • Add Argon gas and seal with parafilm to keep it fresh longer
  3. Aspirate PBS from tube and add 200ul 4% PFA
    • Incubate at 37C for 15min
  4. Wash twice using cold nf-SSPE
  5. Add 0.25% TX-100 in nf-2XSSPE for 5min at RT
  6. Wash with cold nf-H2O twice
  7. Add 200ul 0.01% pepsin in 0.01N HCl (2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O) for 1min at 37C
    • Try 1min, 3min, 5min
  8. Wash with 2ml nf-PBS three times
  9. Prepare 2X Reverse Transcription Mix on ice
Components Volume
H2O 157
10X M-MuLV Buffer 20
25mM dNTP 2
4mM aa-dUTP 2
100uM FISSEQ_RT 5
RNase Inhibitor 2
M-MuLV RTase 10
Total 200
  1. Incubate 10min at 4C and then ~18hr at 37C