User:Karmella Haynes/Notebook/Polycomb project/2010/03/30

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03/30/10

  • ✓ Cytology: Antibody staining & mounting; sign up for Silver scope (tomorrow morning)
  • ✓ KAH128-8: Screened cells for RFP expression (yesterday); expand inducible clones (5 total) in 6-well plate



Antibody Staining > Block: overnight at 4°C (yesterday)
> Primary (R.T./ 2 hr.):
--> Place sheet of parafilm into 24-well lid (labeled). Put 20 μL of ab mix onto parafilm. Invert cover slip cell-side down on droplet. Seal in humidified box.

  1. α-H3 rabbit (ab1791), 1:500
  2. α-H3K27me3 mouse (ab6002), 1:500
  3. α-H3K9me3 rabbit (ab8898, new), 1:500

> Transfer cover slips back into 24-well plate. Wash 4x 5min. PBS
> Secondary (R.T./ 1 hr.):
--> Place sheet of parafilm into 24-well lid (labeled). Put 20 μL of ab mix onto parafilm. Invert cover slip cell-side down on droplet. Seal in humidified box.

  1. goat α-rabbit-Alexaflour488, 1:1000 + Hoescht 33325, 1:1000
  2. goat α-mouse-Alexaflour488, 1:1000 + Hoescht 33325, 1:1000
  3. goat α-rabbit-Alexaflour488, 1:1000 + Hoescht 33325, 1:1000

> Wash 4x 5min. PBS
> Confirm RFP/ FITC on Sorger scope
> Mount w/ vinol; allow to set (in the dark) > overnight


3/31/10

Microscopy 03/31/10

--> Silver lab fluorescence scope, 60x objective
--> α-H3K27me3 (mouse) and α-H3K9m33 (rabbit) showed no signal. Images shown for α-H3 rabbit only.
--> Next: optimize staining for H3K27me3 and H3K9me3.


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