Histochemical localization of wall substances Cellulose and lignin, the major cell wall substances, were localized using histochemical reagents and fluorescent dyes in free-hand transverse sections.
Light microscopic dyes
Toluidine blue ‘O’ (Feder and ‘O’ Brien 1968), safranin, and a natural dye Sappan (Caesalpinia sappan) (Benazir 2000; Ravichandran et al. 2005) were used. The sections were stained with 0.05 % toluidine blue ‘O’ (TBO), in benzoate buffer for 2-3 min, 1 % safranin (1g in 100 ml) for 1-2 minutes, and water extracted sappan dye (10 %) for 15-30 minutes. After removing the excess stain, sections were mounted in water and viewed under light microscope (10 x, 40 x, and 100 x).
Sections were stained with 0.01 % aqueous solution of Calcofluor White M2R for one minute (Hughes and McCully 1975) to localize cellulose. Acridine orange (AO) was used at 0.1% in phosphate buffer at pH 6 for 1 min. (Armstrong 1956) and 0.1 % Coriphosphine ‘O’ for 1 minute (Harris and Oparka 1993). The dyes were used for localization of lignin. All were observed under blue and violet excitations of a fluorescent microscope (Nikon, FMZ 2000) and observed using 10x, 40x, and 100x objectives.
Transmission electron microscopy The procedure described by Rodrigues and Estelita (2003), modified with minor changes, was followed for preparing the specimens for TEM observations and standardized in AIIMS (All India Institute of Medical Sciences, New Delhi, India). The culms from Cyperus pangorei were cut into 2 x 2 mm size. Tissue samples were fixed in a mixture of 2.5 % glutaraldehyde and 2 % paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for 12-18 h at 4oC. After washing in buffer, samples were post-fixed in 1% osmium tetroxide (OsO4) in the same buffer for 2 h at 4oC. Samples were then dehydrated in ascending proportions of acetone, starting from 30% to absolute acetone (about 30 min in each step) at 4oC. Such dehydrated samples were infiltrated in resin – toluene mixture in a vacuum incubator overnight and finally embedded in pure resin, araldite CY 212 (TAAB, UK). Ultra-thin sections (1µm) were cut with an ultramicrotome (ultracut E, Reichert, Austria) using a glass knife, fixed on glass slides, and adhered by applying heat. These sections were then stained with aqueous toluidine blue on a hot plate at 70 oC and observed under a light microscope for general and specific observations of the area and quality of the tissue fixation. For electron microscopic examination, thin sections of grey-silver colour interference (70-80 nm) were cut and mounted onto 300 mesh- copper grids. Sections were stained with alcoholic uranyl acetate and alkaline lead citrate for 12 min in each step, washed gently with double distilled water and observed under a Morgagni 268D Transmission Electron Microscope (TEM) (Fei Company, The Netherlands) at an operating voltage of 80 kV. Some photographs were taken by using a CCD camera with a digital mode (Megaview, Fei Company) attached to the microscope.
Benazir et al. (2010). “Sedge fibers and strands,” BioResources 5(2), 951-967.