Immunofluorescence assays were performed on Technovit 8100 sections following the protocol described by us (El-Tantawy et al., 2013 (link); Solís et al., 2016 (link)). Several rat monoclonal antibodies to pectins with high (JIM7, LM20) and low (JIM5, LM19) level of methylesterification, and to various AGP epitopes (LM2, LM6) (PlantProbes, Leeds, United Kingdom) were used (Supplementary Table 1). Sections were treated as follows: PBS for 1 min; 5% bovine serum albumin (BSA) for 5 min; incubation with the primary antibody to pectins or AGPs for 1 h; washing in PBS, three times, 1 min each; incubation with the secondary antibody (anti-rat IgG-conjugated to Alexa 488, Thermo Fisher Scientific, Rockford, IL, United States) diluted 1/25 in PBS for 45 min, in darkness; washing in PBS, three times, 1 min each; staining with DAPI (1 mg/ml) for DNA; washing in PBS and distilled water. After that, sections were mounted in Mowiol and observed in a confocal microscope (Leica SP5, Leica Microsystems, Wetzlar, Germany). In order to make appropriate comparison among fluorescence signals, the same settings for sample excitation and capture of emission were kept in the confocal microscope for each antibody in all samples. Controls were performed by omitting the primary antibody.
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