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=BA8 Section DARTFISH with Fiducial Beads= *Practice DARTFISH on BA8 tissue sections *Use FluoSpheres Amine-Modified Microspheres 0.2um (505/515) as fiducial markers ==Determine how to incorporate [https://www.lifetechnologies.com/order/catalog/product/F8764 fiducial beads]== *Amount to add, what step to add, for how long *Cross-link with BS(PEG)9 ===MERFISH paper=== *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=== *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=== *Keep dilute, adjust pH, reduce ionic strength *And then bath sonicate ==DARTFISH on BA8== #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''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''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
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