Tamarind (Tamarindus indica L.) seeds were obtained from Jungle Seeds, Watlington, UK) and nasturtium (Tropaeolum majus L. cv Tom Thumb) seeds from Mr. Fothergill's Seeds Ltd., Newmarket, UK. Tamarind and nasturtium seeds were imbibed for 24 h and then pieces of cotyledon parenchyma were excised, fixed and prepared for embedding in LR White resin with subsequent sectioning for indirect immunofluorescence analysis as described previously [8 (link)]. Tobacco (Nicotiana tabacum L.) and pea (Pisum sativum L.) plants were grown in a greenhouse with 16 h days and maintained between 19 and 23°C. Regions of second internodes from the top of six-week old plants were fixed, embedded in wax and sectioned as described previously [46 (link)].
In addition to LM15, three further monoclonal antibodies were used in this study using indirect immunofluorescence: CCRCM1, a mouse monoclonal antibody to a fucosylated epitope of xyloglucan [19 (link)], a gift from Dr. Michael Hahn (CCRC, University of Georgia, USA), JIM5, a rat monoclonal antibody to methyl-esterified and unesterified epitopes of HG [32 (link)] and LM6, a rat monoclonal antibody to arabinan [34 (link)]. Section pre-treatment to remove HG from cell walls involved incubation of sections with a recombinant microbial pectate lyase 10A [47 (link)] (a gift from Prof. Harry Gilbert, University of Newcastle-upon-Tyne) at 10 μg/mL for 2 h at room temperature in 50 mM N-cyclohexyl-3-aminopropane sulfonic acid (CAPS), 2 mM CaCl2 buffer at pH 10 as described [10 (link)]. The high pH of the enzyme buffer removes HG methyl esters in cell walls and results in HG being susceptible to pectate lyase degradation and also suitable for recognition by JIM5. Sections not treated with the pectate lyase were incubated for an equivalent time with the high pH buffer without enzyme and imaged as untreated controls. After enzyme or buffer treatment, sections were incubated in phosphate-buffered saline (PBS) containing 5% (w/v) milk protein (MP/PBS) and a 5-fold dilution of antibody hybridoma supernatant for 1.5 h. Samples were then washed in PBS at least 3 times and incubated with a 100-fold dilution of anti-rat IgG (whole molecule), or anti-mouse IgG, linked to fluorescein isothiocyanate (FITC, Sigma, UK) in MP/PBS for 1.5 h in darkness. The samples were washed in PBS at least 3 times and incubated with Calcofluor White (0.2 μg/mL) (Fluorescent Brightner 28, Sigma, UK) for 5 min in darkness. Samples were washed at least 3 times and then mounted in a glycerol-based anti-fade solution (Citifluor AF1, Agar Scientific, UK). Immunofluorescence was observed with a microscope equipped with epifluorescence irradiation and DIC optics (Olympus BX-61). Images were captured with a Hamamatsu ORCA285 camera and Improvision Volocity software.
In addition to LM15, three further monoclonal antibodies were used in this study using indirect immunofluorescence: CCRCM1, a mouse monoclonal antibody to a fucosylated epitope of xyloglucan [19 (link)], a gift from Dr. Michael Hahn (CCRC, University of Georgia, USA), JIM5, a rat monoclonal antibody to methyl-esterified and unesterified epitopes of HG [32 (link)] and LM6, a rat monoclonal antibody to arabinan [34 (link)]. Section pre-treatment to remove HG from cell walls involved incubation of sections with a recombinant microbial pectate lyase 10A [47 (link)] (a gift from Prof. Harry Gilbert, University of Newcastle-upon-Tyne) at 10 μg/mL for 2 h at room temperature in 50 mM N-cyclohexyl-3-aminopropane sulfonic acid (CAPS), 2 mM CaCl2 buffer at pH 10 as described [10 (link)]. The high pH of the enzyme buffer removes HG methyl esters in cell walls and results in HG being susceptible to pectate lyase degradation and also suitable for recognition by JIM5. Sections not treated with the pectate lyase were incubated for an equivalent time with the high pH buffer without enzyme and imaged as untreated controls. After enzyme or buffer treatment, sections were incubated in phosphate-buffered saline (PBS) containing 5% (w/v) milk protein (MP/PBS) and a 5-fold dilution of antibody hybridoma supernatant for 1.5 h. Samples were then washed in PBS at least 3 times and incubated with a 100-fold dilution of anti-rat IgG (whole molecule), or anti-mouse IgG, linked to fluorescein isothiocyanate (FITC, Sigma, UK) in MP/PBS for 1.5 h in darkness. The samples were washed in PBS at least 3 times and incubated with Calcofluor White (0.2 μg/mL) (Fluorescent Brightner 28, Sigma, UK) for 5 min in darkness. Samples were washed at least 3 times and then mounted in a glycerol-based anti-fade solution (Citifluor AF1, Agar Scientific, UK). Immunofluorescence was observed with a microscope equipped with epifluorescence irradiation and DIC optics (Olympus BX-61). Images were captured with a Hamamatsu ORCA285 camera and Improvision Volocity software.
Free full text: Click here
