GUS Staining of Pollen

(Janine’s protocol)

1. Add 200µl of acetone, vortex briefly.

2. Remove acetone and pipette into flat-bottom 96-well plate (polystyrene)

3. Centrifuge plate at 3000 x g for 3-5 minutes.

4. Invert plate carefully and blot dry with paper towels

5. Add 40µl of X-Gluc stain solution to each well (multi-pipettor) and incubate at 37° C overnight.

<center> <p> <font><b><font>X-Gluc Solution</font> </b> </font></p> </center> 1 20 mg/ml X-Gluc concentration.  e.g. For a 1.5 ml tube (microfuge tube) dissolve 30 mg (.03 g) of X-Gluc in 1.5 ml DMSO (dimethyl sulfoxide).
2 Cover with foil.
3 Vortex well.
4 Keep at -20° C. <center> <p> <b><font>X-Gluc stain solution</font> </b><br /> <i>~undefinedNote: 4ml of stain solution are necessary to stain 1 96-well plate.</i></p> </center>
  <center> <table> <tbody> <tr> <td> <i><b><u>Stock solution (10x)</u> </b> </i> <p> <i>50 mM ferrocyanide<br /> 50 mM ferricyanide </i></p> <p> <i>500mM NaPO_{<font>4</font>} , pH 7 </i></p> <p> <i>20mg/ml X-Gluc (in DMSO) </i></p> <p> <i>ddH_{<font>2</font>} O</i></p> </td> <td> <i><b><u>Working solution (1ml)</u> </b><br />   </i> <p> <i>100µl </i></p> <p> <i>100µl </i></p> <p> <i>25µl </i></p> <p> <i><u>775µl</u> </i></p> <p> <i>1000µl = 1 ml</i></p> </td> </tr> </tbody> </table> </center>

• Cover with foil
•  Keep at 4° C.

<center> <p>  </p> </center>
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