Matt:LabNotes/2015-8-3

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BA8 Section DARTFISH with Fiducial Beads[edit]

  • Practice DARTFISH on BA8 tissue sections
  • Use FluoSpheres Amine-Modified Microspheres 0.2um (505/515) as fiducial markers

Determine how to incorporate fiducial beads[edit]

  • Amount to add, what step to add, for how long
  • Cross-link with BS(PEG)9

MERFISH paper[edit]

  • Uses 0.2um carboxylate-modified fluorescent beads (LifeTech F-8809)
    • 1:1,000 dilution in 2X SSC sonicated for 3min and then incubated for 5 min in sample
    • Then sample washed once with 2X SSC and then fixed with 4% PFA in 2X SSC for 30min

Buffer[edit]

  • Since amine-modified (cationic)
    • Avoid borate, citrate, or phosphate
    • Keep ionic strength of buffer as low as possible due to small microsphere size
    • pH < 9.0
  • Can't use Tris because it has primary amine group that will compete with microspheres for BS(PEG)9 cross-linking?
  • Barbital buffer: 50ml 0.2M sodium barbital (Veronal, 41.2g in 1000ml) add 17.5ml 0.2M HCl and dilute to 200ml with DI
  • Cacodylate buffer: 4.28g/100ml and add 0.2M HCl
    • Good for pH 5.0-7.4
    • Avoids adding phosphates
    • Will not react with aldehyde fixatives (because no amine)
  • Good's Buffers
    • TES
    • HEPES
    • TAPSO
    • POPSO
    • HEPPSO
    • HEPPS

Keeping Monodisperse[edit]

  • Keep dilute, adjust pH, reduce ionic strength
  • And then bath sonicate

DARTFISH on BA8[edit]

  1. Prepare plastic culture dish with hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH sterilize and UV
  2. Thaw 4% PFA made in Feb2015 and stored at -20C
  3. Take out BA8 section from -80C and incubate on 50C hot plate for 3min
  4. Submerge section in 5ml 4% PFA in 6 well culture plate for 15min at 37C
  5. Wash with 2X SSPE by submerging in 2 clean wells of culture plate with 5ml SSPE
  6. Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
  7. Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
    • 0.25% TX-100 in 2XSSPE tube had crack in cap so not completely sealed
  8. Wash with nuclease-free H2O three times
  9. Add 0.01% Pepsin in 0.1N HCl and incubate for 5min at 37C
    • 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
  10. Wash with nuclease-free PBS three times
  11. Prepare Reverse Transcription Mix on ice
Components Volume
H2O 157
10X M-MuLV Buffer 20
25mM dNTP 2
2mM aa-dUTP 4
100uM N9 5
RNase Inhibitor 2
M-MuLV RTase 10
Total 200
  1. Incubate 10min at 4C and then ~15hr at 37C
  • Tissue was completely degraded the next morning
    • First guess is old PFA in -20C was not effective fixative
    • Will try making it from fresh 16% PFA ampules