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Host species: Donkey
C. elegans germline and gonad immunostaining protocol
Carvalho lab (University of Saskatchewan, Canada)
1- Dissect gonads in distilled water and remove excess solution with a 3MM paper.
2- Add a coverslid, immerse slide in liquid nitrogen for 30 sec.
3- “Freeze and crack” using a razor blade.
4- Fix in methanol 100% at -20ºC for 30 min.
5- Wash three times in TBS-T at room temperature for 5’ each.
6- Block in 0.5% BSA in TBS-T for 2 hrs at room temperature.
7- Incubated in 1:500 diluted anti-H3K27me3 (AbCam – ab6147) in Block solution, overnight at 4ºC.
8- Wash three times in TBS-T at room temperature for 5’ each.
9- Incubate in 1:300 diluted anti-mouse IgG FITC-conjugated for 2 hrs at room temperature.
10- Wash three times in TBS-T at room temperature for 5’ each.
11- Incubate in 0.5ug/ul DAPI solution for 10 min at room temperature.
12- Wash in TBS-T for 1 hour.
Submitted Jan 18 2013