11:00 A – 2:00P, 7:00P – 10:45 P
probe making: precipitate, wash, resuspend and freeze probes. Ready to try.
- 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.
- More PBT rinses
- Post fix 30 min in .1% GA, 4% PFA.
- PBT rinses. Back to 4C until ready for STORM imaging (Sunday).
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).