9:15 A – 7:00P, 8:00P – 12:00A
RNA FISH + Immuno-stain in cultured cells (developing protocol:)
- warming up hybe solution for hot washes
- remove, label and save probes (in yellow -20C dilute probes box, labeled as S2).
- 3x Hot wash with hybe solution at 55C
- 3x wash at RT with PBT (loosing more cells / detaching in washes… )
- Block 45 min
- primary antibody incubation, 1.5 hrs, RT
- Wash 2x with PBT
- Block 30 min
- Secondary antibody incubation, 1 hr, RT
- Wash 2x with PBT
- Block 30 min
- Tertiary antibody incubation, 1.5 hr RT
- Wash 3x with PBT
- post fix 4% FA .1% GA 5 min
- test stains on confocal:
- RNA FISH very backgroundy, no nuclear signal / nascent transcripts. Should troubleshoot this protocol using a probe for something that’s more strongly expressed? look up protocols for S2 cells?
DNA FISH+ Immuno-stain in cultured cells (developing protocol:)
- warming up post-hybe. (protocol for cells goes straight to 2x SSCT)
- Remove and save probe (labeled as S2 cent-STORM, in green -20C box)
- Hot wash 1, 15 min at 60C (on heat block).
- RT wash, 15 min in 2x SSCT
- PBT wash 15 min RT. Preping for antibodies
- Block 45 min
- Primary antibody incubation, 1.5 hrs
- Wash 2x with PBT
- Block 30 min
- Secondary antibody incubation, 1 hr, RT
- Wash 2x with PBT
- Block 30 min
- Tertiary antibody incubation, 1.5 hr RT
- Wash 3x with PBT
- post fix 4% FA .1% GA 5 min
- Check stains on confocal, all look good
Embryos for STORM
- Save H3-750 1:50 in 15 mL tube, (in -20C ,my 15 mL tube rack)
- PBT rinses
- mount subset of embryos for confocal
- dehydrate into ethanol
- embed in resin
Embryo DNA-FISH troubleshooting fix
- post fixed all embryos with conventional fix (4% FA, .1% GA, 1hr).
- separate 1 tube for troubleshooting where the dye is lost (standard treatment)
- 1 tube 9% FA for another 1 hr. split in half –> 9% FA O/N at 4C.
- 1 tube 9% FA, 3% GA for another 1 hr, split in half –> 9% FA, 3%GA O/N at 4C.
- No stain after ~2 hrs of EtOH dehydration in conventional tube.
Stocks
- Check flies
BIRS
- start coverletter for NSF
- draft agenda
- draft main sections of proposal
- send to co-organizers for input