Plant Pollen Tube Staining Solution (Aniline Blue Fluorescence Method)

Cat. No.: P1511521

GRADE & PURITY BioReagent, Biological Stain, for Fluorescence analysis, for Microscopy

Overview

  Flowering plants need to transport sperm cells to the ovule via pollen to complete double fertilization. This process must overcome two key obstacles: first, the pollination process from pollen release from the anther to reaching the pistil, which in bisexual flowers relies on anther dehiscence, while in unisexual flowers requires assistance from wind or insects; second, after pollen adheres to the stigma, sperm cells must be transported to the egg cell enclosed within the ovule. To achieve this, pollen germinates a pollen tube to facilitate the directional transport of sperm cells. Its growth mainly includes stages such as pollen-stigma adhesion, pollen hydration, pollen tube germination and elongation, micropyle-guided growth, and sperm cell release.

  Callose is a polysaccharide formed by glucose residues linked in a non-cellulosic manner. It participates in important biological activities such as plant sieve tube metabolism and gametophyte development. Its synthesis and degradation directly affect normal plant growth and metabolism, making it a significant subject in plant research.

  Plant Pollen Tube Staining Solution (Aniline Blue Fluorescence Method) contains aniline blue staining solution and a diluent. The principle is based on water-soluble aniline blue specifically binding to callose in pollen tubes, which emits yellow-green fluorescence upon ultraviolet excitation. This product is simple and quick to operate, suitable for fluorescence microscope observation of pollen tube growth in plants such as rice and Arabidopsis. It is for research use only and not intended for clinical diagnosis or other non-research purposes.

Materials to Be Prepared by User

• AAF Fixative Solution (50%) or Carnoy's Fixative Solution or AF Fixative Solution, 50% Ethanol, Absolute Ethanol, 1× PBS, Glycerol-Gelatin Mounting Medium or Anti-Fade Mounting Medium
• Fresh plant tissue sections (pollen tubes)

Procedure (For Reference Only; Fixation and Softening Methods May Vary Depending on Actual Conditions)

1. Prepare the Aniline Blue Working Solution: Mix the Aniline Blue Staining Solution and Aniline Blue Diluent in equal proportions. Prepare immediately before use.

2. Take fresh pistils and fix them in AAF Fixative Solution (50%), Carnoy's Fixative Solution, or AF Fixative Solution for 10 hours. Rinse thoroughly with distilled water.

3. Soften the samples in 2 mol/L NaOH at 45°C in a water bath for 2 hours to overnight. Rinse three times with distilled water.

4. Stain with the Aniline Blue Working Solution at room temperature, protected from light, for 4 hours.

5. Place the stained pistils (longitudinally including the ovary) on a glass slide. Add 25 μL of aqueous Glycerol-Gelatin Mounting Medium or Anti-Fade Mounting Medium, cover with a coverslip, and gently flatten.

6. Observe the pollen tubes in the style under a fluorescence microscope using UV excitation light (excitation wavelength 330~385 nm (BP), emission wavelength 425 nm (LP)). Use unstained pollen tubes as a control.

Staining Results

  Callose produced by pollen tubes emits yellow-green fluorescence under UV excitation, with no background staining.

Notes

1. When conditions permit, it is recommended to subject the plant tissue immersed in fixative to negative pressure/vacuum treatment for 20 minutes to aid fixative penetration.

2. If the experiment is not to be performed immediately or a large number of samples need to be processed simultaneously, samples stored in 100% ethanol can be kept at 2-8°C for at least one week.

3. Photograph the stained samples as soon as possible after staining to preserve the results.

4. The active components of the staining solution are prone to decomposition. It is recommended to prepare the working solution immediately before use and use it within 3 hours. A slight color fading of the reagent during use is a normal phenomenon.

5. Fluorescent staining is susceptible to photobleaching. Take care to avoid ambient light other than the excitation light during staining, transfer, and observation.

6. Use the reagents as soon as possible after opening to avoid affecting subsequent experimental results.

7. For your safety and health, please wear a lab coat and disposable gloves during operation.

Storage and Shipping

Storage

Protected from light,Room temperature

Shipped In

Normal

Stability And Storage

Store at room temperature long term (12 months). Store in the dark.

Contents & Storage

P1511521	Component	2×25 mL	Storage
P1511521A	Aniline Blue Staining Solution	25 mL	RT. Store in the dark.
P1511521B	Aniline Blue Diluent	25 mL	RT. Store in the dark.