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

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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.

  1. H3K27me3 07-449, 1:200
  2. 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

Microscopy 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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