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

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04/01/10

  • ✓ 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.

  • goat anti-rabbit-Alexafluor488, 1:1000; Hoescht, 1:1000
  • goat anti-mouse-Alexafluor488, 1:1000; Hoescht, 1:1000

--> Wash w/ 1x PBS, 4x/R.T./5 min.


> 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)

H3me IF optimization
> 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!
Cool DAPI images of different stages of mitosis



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