Matt:LabNotes/2015-12-14

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FISSEQ on Mouse Embryo Test 2[edit]

  • Previous Test
  • Try 2 samples: slide7 (16um thickness) and slide8 (20um thickness)

Embryo Section Info[edit]

  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[edit]

  • 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[edit]

  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

Day 2[edit]

  1. Wash with 1X PBS twice
  2. Add 200ul BS(PEG)9 (20ul BS(PEG)9 stock + 980ul 1X PBS) and incubate 1hr at RT
  3. Wash with 1X PBS twice
  4. Quench with 1M Tris for 30min at RT
  5. Wash with 1X PBS twice
  6. RNA Removal
    • Add 200ul RNase Mix (100ul RNase H 10X Buffer + 10ul Riboshredder + 50ul RNase H + 840ul H2O) and incubate 1hr at 37C
  7. Wash with H2O twice
  8. Add 200ul CircLigaseII mix and incuabate 3hr at 60C
Component Volume
DEPC-H2O 640
CircLigase Buffer 10X 100
MnCl2 50mM 50
Betaine 5M 200
CircLigase II (100U/ul) 10
Total 1000
  1. Wash with 1X PBS twice
  2. Add 200ul 0.5uM FISSEQ_RCA (5ul 100uM FISSEQ_RCA + 995ul 2X SSC + 30% formamide) and incubate 1hr at 60C
  3. Wash with 2X SSC + 30% formamide for 10min at 60C
  4. wash with 2X SSC, 1X SSC, 1X PBS once each
  5. Add 200ul RCA mix and incubate for 15hr at 30C
Component Volume
H2O 174
10X Phi29 Buffer 20
25mM dNTP 2
4mM aa-dUTP 2
Phi 29 (low conc) 2
Total 200

Day 3[edit]

  1. Wash with 1X PBS once
  2. Add 200ul BS(PEG)9 (20ul BS(PEG)9 stock + 980ul 1X PBS) and incubate 1hr at RT
  3. Wash with 1X PBS twice
  4. Quench with 1M Tris for 30min at RT
  5. Wash with 1X PBS twice
  6. Add 200ul 75C preheated 0.5uM FISSEQ_Adpt (Cy3) in 2X SSC + 30% formamide and incubate 10min at RT
  7. Wash with 2X SSC twice
  8. Image with Confocal

Image Results[edit]

  • 20X 0.75 NA

A7 16um[edit]

File:MAX A7 16um Section1.jpg File:Composite A7 16um Section1.jpg

A8 20um[edit]

Section 1[edit]

File:MAX A8 20um Section1.jpg File:Composite A8 20um Section1.jpg

Section 2[edit]

File:MAX A8 20um Section2.jpg File:Composite A8 20um Section2.jpg