Matt:LabNotes/2015-12-14

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Revision as of 03:54, 15 December 2015 by >Mzcai (→‎Day 1)
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FISSEQ on Mouse Embryo Test 2

  • Previous Test
  • Try 2 samples: slide2 (20um thickness) and slide3 (25um thickness)

Embryo Section Info

  1. E7.5#3 slide1 is 16um (8 sections beginning of embryo + placenta) on the coverslip there is written A1, B1, C1, D1 to help for the orientation.
  2. E7.5#3 slide2 is 20um (8 sections beginning of embryo + placenta) on the coverslip there is written A2, B2, C2, D2 to help for the orientation.
  3. E7.5#3 slide3 is 25um (7 sections beginning of embryo + placenta) on the coverslip there is written A3, B3, C3, D3 to help for the orientation.
  4. E7.5#3 slide4 is 16um (7 sections mainly embryo) on the coverslip there is written A4, B4, C4, D4 to help for the orientation.
  5. Used: E7.5#3 slide5 is 20um (7 sections middle embryo) on the coverslip there is written A5, B5, C5, D5 to help for the orientation.
  6. Used: E7.5#3 slide6 is 25um (7 sections middle embryo) on the coverslip there is written A6, B6, C6, D6 to help for the orientation.
  7. E7.5#3 slide7 is 16um (10 sections middle embryo) on the coverslip there is written A7, B7, C7, D7 to help for the orientation.
  8. E7.5#3 slide8 is 20um (12 sections middle/end embryo) on the coverslip there is written A8, B8, C8, D8 to help for the orientation.
  9. E7.5#3 slide9 is 25um (6 sections end embryo+ adult liver) on the coverslip there is written A9, B9, C9, D9 to help for the orientation.

Protocol

  • Previously did 0.01% Pepsin for 10min at RT and had too much tissue loss
  • Try 0.01% Pepsin for 1-2min at 37C

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 18mm diameter hole (16mm adhesive hole)
    • When peeling off scotch tape from coverslip (that was used to tape coverslips to glass slides), it peeled off a clear film as well...
    • What kind of clear film would develop on coverslip at -80C?
    • Use Double-sided tape: Adhesives Research IS-8458-19
  3. Wash tissue section twice using cold nf-SSPE for 5min each
    • A8 leaked so used 200 Gap filling glue
  4. Add 0.25% TX-100 in nf-2XSSPE for 5min at RT
  5. Wash with cold nf-H2O twice
  6. Add 200ul 0.01% pepsin in 0.01N HCl (2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O) for 1min at 37C
  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