11:00 A – 2:00P, 7:00P – 10:45 P
probe making: precipitate, wash, resuspend and freeze probes. Ready to try.
Cryo-section staining
- secondary stain and wash of DFISH coverslips
- No sections remaining on DFISH coverslips. Try crosslinking?
- staining on coverglass does lead to substantial background from antibodies stuck to coverglass. Presumably they sample covers the glass so this shouldn’t affect sample background?
- Antibody stain: stain with H3K27me3-647. No strong nuclei labeling, dots in corners of some nuclei.
- Dehydration of sample seems to be serious problem. Tested one sample labeled with DAPI. As section dried, DAPI stain disappears. Rehyrdration restores stain to some cells but many have a hollow, sucked out middle, and sometimes a bright foci in the edge of ‘nucleus’–(as defined by the cytoplasmic background scattering. These are similar to how the H3K27me3-647 spots looked. Rehydrated this sample (red band) anyway and comparing staining to hydrated version (pink band).
- Round 2
- Step 1, add Hoechst to frozen sucrose, visualize sections/confirm quality (take image to compare to later)
- Try crosslinking sections to coverglass with brief PFA treatment (10 min in DFISH fix mix)
- Incubating red and pink coverslips in petri dishes. Staining in 5 mL DFISH hybe (should be able to reuse), with cent-probe at 1:500 (fortunately I have loads of this still). We’ll work on the downsized approach for smaller volumes later.
- Coverslip 3: fix 10 min (DFISH mix). Rinse in PBT. Block 1 hr. Primaries: rb a-PC + m-Dm01 (reused from frozen 1x use 10/22). incubating O/N at 4C.
Cell staining
- More PBT rinses
- Post fix 30 min in .1% GA, 4% PFA.
- PBT rinses. Back to 4C until ready for STORM imaging (Sunday).
Cell culture — added Penacillin/Streptomycin (50 uL stock in 50 mL solution) to S2 cell culture. Changed medium and diluted cells. Culture bottle has some precipitates, should move to fresh bottle next week.
antibodies to test soon: H3K9me2, H3K27me3-647 direct label, rb 750s
BIRS: write to Dalya and Jim Sweeney.
Order sigma-coat (for polytene squashes), tweezers (for slide manipulation in petri dishes), 1-Butanol (for probe making).