Lignin Staining Solution (Safranin Method)

Guidelines (The following is our recommended protocol, which serves only as a guideline and should be modified according to your specific needs).
(I) Staining for Lignin Distribution in Woody Plant Branches
1. Suitable materials: Transverse sections of annual branches of woody plants (fixed with FAA, sectioned to 25 μm using a sliding microtome).
2. Immerse the sections in 2% Safranin O aqueous solution and stain at room temperature for 2 h (or stain overnight to enhance fluorescence signals).
3. Wash away excess staining solution with distilled water.
4. For double staining to distinguish cellulose, counterstain with Fast Green FCF or Astra Blue for 15 seconds to 3 min, followed by rapid dehydration with ethanol.
5. Dehydrate, clear, and mount the sections.
6. Observe under a bright-field or epifluorescence microscope.
7. Staining results: Lignified cell walls (vessels, fibers) appear pink to red, with the color intensity positively correlated with the degree of lignification; under fluorescence, vessel walls, cell corners, and intercellular layers show red fluorescence, while secondary walls of fibers show blue-purple fluorescence.
(II) In Situ Quantitative Staining for Lignin in Herbaceous Plant Stems
1. Suitable materials: Inflorescence stems of *Arabidopsis thaliana*, flax stems, etc., embedded in 4% agarose and sectioned to 80 μm using a vibratome.
2. Place the sections in a 48-well plate, add 0.2% Safranin O, and stain on a shaker at room temperature in the dark for 10 min.
3. Aspirate the staining solution, replace with 50% ethanol, and wash in the dark for 10 min.
4. Rinse twice with ultrapure water, 15 min each time.
5. Mount the sections in water.
6. Acquire 3-channel images using a confocal microscope (405 nm autofluorescence, 488 nm/530-560 nm green channel, 561 nm/570-600 nm red channel) with consistent parameters.
7. Perform FIJI ratio analysis (570-600 nm / 530-560 nm), and automatically segment three regions (cell corners, intercellular layers, secondary walls) to quantify lignin.
8. Staining results: Regions with high lignin content (xylem) show high ratio values (reddish), while regions with low lignin content (cambium, phloem fibers) show low ratio values (purplish), enabling statistical comparison between wild-type and mutant samples.
Lignin Staining Solution (Safranin Method) (0.2% (w/v); 10 min) enables ratiometric fluorescence quantification and automatic segmentation of lignin in the cell corners, compound middle lamellae, and secondary cell wall regions of Arabidopsis thaliana inflorescence stem cross-sections, and detectable differences in lignin content exist between wild-type and mutant plants^[1].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.