2.3 Evaluation of the internal leaf anatomy: idioblast characterization
To better understand the structure of the idioblasts in the mesophyll, hand-cut transverse thin sections (perpendicular to the leaf surface) were made and stained with a solution of 0.01% w/v calcofluor white in 10mM CHES buffer with 100mM KCl (pH=10) (Hughes & McCully, 1975), to identify cellulose-rich walls, and then with Auramine-O in 0.05 M Tris/HCl buffer, (pH=7.2), to detect cutinized lipids (Heslop-Harrison & Shivanna, 1977). These sections were visualized with a Zeiss Axioskop microscope equipped with epifluorescence, and imaged with an AxioCam HRc camera, operated by Zen Blue software with multichannel wavelength detection. For calcofluor white, we used a DAPI narrow (Zeiss 48702) excitation G 365, bandpass 12 nm; dichroic mirror FT 395; barrier filter LP397; for auramine O, we used an AF488/FITC/GFP (Zeiss Filter set 9) excitation 450-490; dichroic mirror 510; emission filter LP515. We isolated the idioblasts by macerating 1 mm2 leaf pieces in a solution containing acetic acid: hydrogen peroxide 1:1 (v/v) at 60ºC for 2 days. The debris of these samples was then mounted in distilled water or glycerol and visualized with a Zeiss LSM700 Confocal Microscope. 3D images were obtained without staining with the Objective: 63x/1.40 Oil DIC M27 Plan-Apochromat.
Samples obtained from five previously fixed leaves in FAA were washed in distilled water three times, 1h each, and dehydrated with an increasing gradient of aqueous ethanol concentrations (10%, 30%, 50%, 70%, 100% (x3)). They were then soaked in the infiltration solution of Technovit7100 (Electron Microscopy Sciences, Hetfield, PA, USA) for several weeks, and polymerized under anoxic conditions. Blocks with samples oriented parallel to the leaf surface (paradermal) were serially sectioned at 4 µm with a Leica RM2155 rotary microtome (Leica Microsystems, Wetlar, Germany). Sections were then stained with an aqueous solution of 0.025% toluidine blue for general structure of the leaf tissues (Feder & O’Brien, 1968), and finally imaged with a Zeiss AxioImager A2 Upright microscope equipped with an AxioCam 512 camera and Zen Blue Pro with multichannel software.