Phluoroglucinol

The experiment was carried out on green parts of the basal sections of the stalks grown in the field. From each line and the control 5 plants were taken to analysis.
Gently sliced into pieces, the cross-sections were transferred onto watchglass and stained with phluoroglucinol (Sigma-Aldrich)-HCl(12 N) solution for 5 minutes. Fresh specimens were probed under Axio.Scope A1 microscope (Carl Zeiss) equipped with AxioVs40 v4.8.2.0 software.

Approx. 1 cm long pieces of 70% ethanol-fixed internodes harvested at the ripening stage of growth were dissected, rehydrated and embedded in 8% agarose in Eppendorf tubes. The blocks were glued on a metallic holder and 100 μm thick sections were generated using the vibratome Leica VT1000 s. The sections were collected in PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.7 mM KH₂PO₄, pH 7.5) in small sieves in 24-microwell plate. The sections were blocked by 5% milk in PBS for 30 min, incubated for 1 h with the primary antibody (LM12, PlantProbes) at room temperature in a solution of 5% milk in PBS at the 1:10 dilution, washed two times with PBS and incubated for 1 h in anti-rat secondary antibody conjugated with AlexaFluor488 (Invitrogen) at 1:500 dilution. The sections were then washed two times with PBS stained with Calcofluor (0.1 mg mL⁻¹ in PBS), washed and mounted on the glass slide in CITIFLUOR (Agar Scientific) mounting media. The sections were scanned using a Leica SP5 laser scanning confocal microscope (LSCM) equipped with Ar and HeNe lasers. All sections were scanned with the same settings.
The saturated solution of phluoroglucinol (Sigma) in 20% HCl was used to stain lignin (Wiesner stain). The sections were placed in the drop of solution, incubated app. 5 min and directly observed using a Olympus BX41 light microscope.