04/04/10
- ✓ 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.
- H3K27me3 07-449, 1:200
- H3K9me3 8898 (old), 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
--> 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)
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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