Chromatin Experiments To Do

in situs

BX-C

• Expression and DNA locus multiplex
  1. whole-mount embryo in situs for AbdB, AbdA, and Ubx with dig probes anti-dig-405,647
  2. ultra cryo section embryos.
  3. label with WGA-488 on coverlgass, then STORM the RNA labels.
  4. DNA-FISH BX-C or individual AbdB, AbdA and Ubx loci

Y expression state

• Yprobe + RNase + denature
• Yprobe – RNase – denature
• Yprobe – RNase + denature
• Yprobe – RNase – denature; Image; Yprobe + RNase + denature; Image (RNA / DNA double, same locus).

Methods

Troubleshooting embryo in situs

• sections still get lost / detach
• direct contact with glass may remove sections?
• try with very thin PDMS spacers? (can we even make these?)
• try poly-lysine coating and post-fix of sections. (I believe I have had partial success with post-fix previously).

Probe building

• Order T7 primers to make antisense RNA probes with tails for AbdB, AbdA and Ubx
• Synthesize RNA and test in whole mount traditional RNA hybe protocol (I hope the tails stick!)
• Try 3+ on AbdA AbdB Ubx probe making