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

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04/04/10

 * &#x2713; Cytology: Fix & stain KAH126-1, 130-2, 132-8

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

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

> Primary stain (rabbit polyclonal) --> "Face down" method (see 3/30/10); 20 μL diluted ab; R.T./2.5 hrs. --> Wash w/ 1x PBS, 4x/R.T./5 min.
 * 1) H3K27me3 07-449, 1:200
 * 2) H3K9me3 8898 (old), 1:400

> 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

> 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)

4/05/10



--> Very nice foci in 130-2. Process images for red/green overlay in Photoshop; make Powerpoint data slide.


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