Matt:LabNotes/2015-10-7

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FISSEQ on Mouse Embryo Test[edit]

  • Try 2 samples: slide5 (20um thickness) and slide6 (25um 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. 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. 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]

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 16mm diameter hole
    • Use Double-sided tape: Adhesives Research IS-8458-19 (Hosuk originally mistakenly told me 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

Day 2[edit]

  1. Wash with 1X PBS twice
    • Dishes had small leak so had to seal with gap-filling glue
  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]

slide 5 20um[edit]

  • Section 1 10X

File:Cropped BF 20151009 E7.5 a5 20um 10X Section1.jpg

  • Section 2 10X

File:Cropped BF 20151009 E7.5 a5 20um 10X Section2.jpg

  • Section 1 20X

File:MAX 20151009 E7.5 a5 20um FISSEQ 20X Cy3.jpg File:Merge MAXandBF 20151009 E7.5 a5 20um FISSEQ 20X Cy3.jpg

  • Section 1 63X

File:MAX 20151009 E7.5 a5 20um FISSEQ 63X Cy3.jpg File:Merge MAXandBF 20151009 E7.5 a5 20um FISSEQ 63X Cy3.jpg

  • Section 2 had no fluorescence in Cy3 channel for no apparent reason

slide 6 25um[edit]

  • 10X

File:Cropped BF 20151009 E7.5 a6 25um 10X.jpg

  • 63X

File:MAX 20151009 E7.5 a6 25um FISSEQ 63X Pos2 Cy3.jpg File:Merge MAXandBF 20151009 E7.5 a6 25um FISSEQ 63X Pos2 Cy3.jpg

FISSEQ on BA8 for Harvard[edit]

  • 1 slide with 2 sections and 1 coverslip

Day 1[edit]

  1. Prepare 1 plastic culture dishes with hole, tweezers, large plastic dish, slide gasket, and 50C hot plate
    • EtOH and RNaseZap sterilize and rinse with nf-H2O
  2. Made 40ml 4% PFA at RT
    • 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nf-H2O
  3. Take out BA8 section (1 glass slide1 and 1 coverslip1) from -80C and incubate on 50C hot plate for 3min
  4. Submerge coverslip1 in 5ml 4% PFA in 6 well culture plate for 15min at 37C
  5. Submerge glass slides in 35ml 4%PFA in 50ml tube for 15min at 37C
  6. Wash twice with 2X SSPE by submerging
  7. Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
  8. Aspirate any liquid and attach gaskets to glass slides
  9. Add 0.25% TX-100 in 2XSSPE and incubate 5min at RT
  10. Wash with nuclease-free H2O three times
  11. Add 0.01% Pepsin in 0.01N HCl and incubate for 5min at 37C
    • 2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O
  12. Wash with nuclease-free PBS three times
  13. Prepare 3X Reverse Transcription Mix on ice
Components Volume
H2O 477
10X M-MuLV Buffer 60
25mM dNTP 6
4mM aa-dUTP 6
100uM N9 or 100uM FISSEQ_RT 15
RNase Inhibitor 6
M-MuLV RTase 30
Total 600
  1. Incubate 10min at 4C and then ~13hr at 37C
    • Put slide in 50ml tube with wet towel and para film around cap
    • Parafilm dish and put in humidified bag with mouse E7.5

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]

63X[edit]

File:MAX 20151009 BA8 FISSEQ 63X Pos1.jpg

File:Merge MAXandBF 20151009 BA8 FISSEQ 63X Pos1.jpg

Conclusion and Take aways[edit]

  • FISSEQ rolonies in BA8 less than usual
    • Guessing "home made" MnCl2 from dry crystal not good enough, will order 50mM solution from Trevigen
      • CircLigase II epicentre kit is on back order so can't wait for that
  • Significant degradation of mouse embryo sections
    • Try pipetting solutions to side of tissue instead of dropping on top
    • Try fixing tissue again after permeabilization
  • Based on z-start and z-end positions there does not seem to be a difference in tissue thickness between 20um section and 25um section
    • Both measured to be ~15um
    • Is there some maximum tissue thickness that can withstand degradation?
    • Maybe tissue thickness isn't being measured but just where rolonies are
  • Weird spots in mouse embryo tissue (looks like precipitate?)
    • They do not appear in BA8 tissue
    • They do not overlap with rolonies