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)

File:KAH 040110 micro1.tif
> 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!
File:KAH 040110 micro2.tif