9:45 A – 7:30 – 11:55P
Troubleshooting antibodies with polytene
- H3K9me2 failed completely on polytene cells. Dm01 control labeled in parallel looks bright and clean (better than direct labeled Dm01).
- Polytene labeling today: testing H3K27me3 (see if polytene chromosomes label appropriately for this mark. This antibody works at least, not fully convinced the H3K9me2 works at all).
- Try HP1a, the DHSB HP1a gives clean, correct labeling in polytene cells (reference).
- Hot washes for centromere labeled probes. Glass also looks pretty dry. Should do these in situ reactions under coverglass methinks.
- Polytene centromere stain still worked, slide “1” also has good polytene cells. Now blocking.
Image analysis
- En Epc-tou analysis — new version of script.
Antibody research
- This abcam antibody is used in the Riddle papers (ModENCODE), gives good S2 and polytene staining as well as ChIP. (Abcam ab1220, m-H3K9me2). paper1, paper2. Ordered today, hopefully will arrive tomorrow according to Abcam.
- Presumably the rabbit H3K9me2 from Millipore is the Upstate antibody cited by the Johansen lab paper. (why this company refers to itself as Upstate in its contract material and Millipore in its website name is very confusing, but searching for upstate antibodies always returns millipore. Papers are also getting very bad about indicating primary antibody sources clearly — product numbers should accompany commercial antibodies).
- Hp1a works, H3K9me2 of Millipore does not.
Cell culture
- centromere label new S2 cells to test new Abcam H3K9me2 antibody tomorrow
- compare to HP1a in separate stain.
Discussion with Ajaz (see post);
Prep for sectioning
- Rinse some of the recently poly-lysine coated slides
- Hydrate MTDxSCM for sectioning (test Pc organization). Ph antibody is supposed to be very good.
Confocal
- Polytene: K27me3 stains, but rb-488 has strong background. Try different rabbit / redder-dye?
- Polytene 2: almost all cells detached with coverslip after last check on the confocal. Not sure why. No cy5 labeled fragments left, only 1 partial nuclei remaining. Some HP1a visible.
- Dfd, tub / inv,en,Epc,tou in situs all failed: Added dmO with anti-m secondary. foolish.
- Old DNP probes don’t work. Should make new DNP probes, (and clone en inv introns).
- Canceled additional confocal time scheduled to image this data set.
# Call Bender discuss model