User:Karmella Haynes/Notebook/Polycomb project/2010/04/01

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04/01/10

 * &#x2713; Cytology: Fix, permeabilize, and stain U2OS plain

Cytology optimization > Fix & permeabilize --> Wash 1x w/ R.T. PBS --> Fix: 4% formaldehyde/PBS --> Permabilization: 12 wells 5% Triton/PBS; 12 wells cold methanol (R.T./ 10 min.) --> Wash 1x w/ R.T. PBS

> Block: 5% NHS/PBS, R.T./2 hrs.

> Primary stain (rabbit polyclonal, except for 6002) --> "Face down" method (see 3/30/10); 20 μL diluted ab; R.T./2.5 hrs. 1-3. 5% Triton, H3K27me3 07-449, 1:100, 1:200, 1:400 4-6. 5% Triton, H3K27me3 6002 mouse, 1:100, 1:200, 1:400 7-9. 5% Triton, H3K9me3 8898 (old), 1:100, 1:200, 1:400 10-12. 5% Triton, H3K9me3 8898 (new), 1:100, 1:200, 1:400 13-15. methanol, H3K27me3 07-449, 1:100, 1:200, 1:400 16-18. methanol, H3K27me3 6002 mouse, 1:100, 1:200, 1:400 19-21. methanol, H3K9me3 8898 (old), 1:100, 1:200, 1:400 22-24. methanol, H3K9me3 8898 (new), 1:100, 1:200, 1:400 --> Wash w/ 1x PBS, 4x/R.T./5 min.

> Secondary stain --> "Face down" method (see 3/30/10); 25 μL diluted ab; R.T./1 hr. --> Wash w/ 1x PBS, 4x/R.T./5 min.
 * goat anti-rabbit-Alexafluor488, 1:1000; Hoescht, 1:1000
 * goat anti-mouse-Alexafluor488, 1:1000; Hoescht, 1:1000

> Quick check for FITC signal on Sorger's scope --> Looks good! Triton-permeabilized samples look better than methanol-perm. --> ab6002 showed no signal

> Mount Triton-perm samples w/ Vinol solution; allow to set > overnight (in dark)

> Conclusions: --> H3K27me3: use 07-449, 1:200 --> H3K9me3: use 8898 (old), 1:400

--> Appears that cells were happily dividing on the cover slips at the time of fixation. Managed to capture some cool DAPI images of mitosis. Beautiful!


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