Matt:LabNotes/2015-10-7: Difference between revisions

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=FISSEQ on Mouse Embryo Test=
=FISSEQ on Mouse Embryo Test=
*Try 2 samples: 20um and 26um thickness
*Try 2 samples: A5 (20um thickness) and A6 (26um thickness)


==Protocol==
==Protocol==

Revision as of 01:23, 8 October 2015

FISSEQ on Mouse Embryo Test

  • Try 2 samples: A5 (20um thickness) and A6 (26um thickness)

Protocol

Day 1

  1. Sterilize culture dishes and tweezers with EtOH, RNaseZap, and then rinse with nf-H2O
  2. Attach 22mm x 22mm glass coverslips (with mounted sections) to bottom of culture dishes with 16mm diameter hole
    • Use Double-sided tape: Adhesives Research IS-7876-35
  3. Wash tissue section twice using cold nf-H2O for 5min each
  4. Add 0.25% TX-100 in nf-H2O for 5min at RT
  5. Wash with cold nf-H2O twice
  6. Add 200ul 0.01% pepsin in 0.01N HCl (2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O) for 10min at RT
  7. Wash with 2ml of cold nf-PBS three times
  8. 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
    • Parafilm each dish and then put in plastic bag with wet tissue

FISSEQ on BA8 for Harvard