Aniline Blue Staining of Pollen/Pollen Tubes花粉管染色

Materials

Fixative: 10% Acetic Acid in ethanol
1M NaOH
50 mM KPO₄ buffer, pH 7.5:
4.17 mL 1M K₂HPO₄
0.83 mL 1M KH₂PO₄
0.01% aniline blue in 50 mM KPO₄ buffer (dye)
KPO₄ buffer made with 50% glycerol (mounting media)

Method

1.Submerge pistil tissue in about 250µL acetic acid fix for 1.5 hours or more. An eppendorf tube works well for this. Tissue can be left in fix ocernight or longer if necessary.
2. Soften tissue by submerging tissue in 1 M NaOH overnight.
3. Wash 3 times with 50 mM KPO₄ buffer. Be very gentle with the tissue because it is fragile at this stage.
4. Stain with 200µL aniline blue for 5-10 minutes.
5. Transfer to slide, add mounting media, and observe under UV. Squash if necessary.