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]
- Prepare plastic culture dish with hole, tweezers, large plastic dish, and 50C hot plate
- EtOH sterilize and UV
- Thaw 4% PFA made in Feb2015 and stored at -20C
- Take out BA8 section from -80C and incubate on 50C hot plate for 3min
- Submerge section in 5ml 4% PFA in 6 well culture plate for 15min at 37C
- Wash with 2X SSPE by submerging in 2 clean wells of culture plate with 5ml SSPE
- Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
- 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
- Wash with nuclease-free H2O three times
- Add 0.01% Pepsin in 0.1N HCl and incubate for 5min at 37C
- 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
- Wash with nuclease-free PBS three times
- 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 |
- 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