Alexander staining of Arabidopsis anthers

Alexander staining of Arabidopsis anthers

According to Alexander, 1969.

“Differential staining of aborted and non-aborted pollen”

Preparation of the staining solution:

Staining procedure:

1. Select flowers of the desired stage (e.g. flower stage 12 according to Smyth et al., 1990) 

2. Dissect a flower to obtain anthers and mount the anthers onto a microscope slide. It is important that the anthers lay in a good orientation for microscopy, the filament can be squeezed to make it stick to the microscope slide. 

3. Add 20 µl of staining solution and cover the anthers with a 20 mm X 20 mm cover slip. 

4. Seal the edges of the coverslip with Fixogum (rubber adhesive) ^{Note 2}

5. Keep the samples at room temperature (under a fume hood) for at least 2h before imaging ^{Note 3}

6. Image anthers for example with a Zeiss Axio Imager M1 microscope.

Notes:

1. To stain free pollen without anthers the amount of glacial acetic acid and the staining time can be lowered (see Alexander, 1969). 

2. Sealing of the coverslip is optional but highly recommended to contain the toxic staining solution.

3. One has to experimentally determine the correct incubation time based on desired intensity of staining.

References:

Alexander MP. 1969. Differential staining of aborted and nonaborted pollen. Biotech Histochem 44:117–122. doi:10.3109/10520296909063335

Smyth DR, Bowman JL, Meyerowitz EM. 1990. Early flower development in Arabidopsis. Plant Cell 2:755–767. doi:10.1105/tpc.2.8.755