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

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 * style="background-color: #F2F2F2" align="center"|  |Main project page


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03/30/10

 * &#x2713; Cytology: Antibody staining & mounting; sign up for Silver scope (tomorrow morning)
 * &#x2713; 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. > 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. > Wash 4x 5min. PBS > Confirm RFP/ FITC on Sorger scope > Mount w/ vinol; allow to set (in the dark) > overnight
 * 1) α-H3 rabbit (ab1791), 1:500
 * 2) α-H3K27me3 mouse (ab6002), 1:500
 * 3) α-H3K9me3 rabbit (ab8898, new), 1:500
 * 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

3/31/10



--> 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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