Fluoview v 5.0

All observations were carried out and photographs were taken using a confocal laser scanning microscope (CLSM) Olympus BX51 equipped with corresponding software FluoView v. 5.0. (Olympus Corporation, Tokyo, Japan). Representative image sets were selected and edited using the CorelDrawX6 graphics program.
Optical signal density was calculated as the ratio of the total sum of values of pixels from the green channel (fluorescence indicating the presence of the antibody) within the selected region of interest to the pixel area of this region. The regions of interest had rectangular shapes and were selected manually to cover the tissue area below the epidermal layer of cells. CLSM data was analysed using a script written in Matlab R2010a (MathWorks, USA). The data were statistically analyzed using OriginPro 8.5 software (Origin Lab v8.5 Pro, Northampton, USA). For comparisons of the mean values, an analysis of variance (one-way ANOVA) followed by post hoc Tukey’s honestly significant difference test was used. For all analyses, the significance level was estimated at p < 0.05.

Immunofluorescence imaging of AGP epitopes at the tissue/cellular level facilitates evaluation of changes in the distribution of AGPs in fruit at different ripening stages. The experiment was conducted using JIM13, LM2, and LM14 antibodies. Semi-thin sections on poly-L-lysine coated glass slides were circled with a liquid blocker Dako-Pen (Sigma Aldrich, USA). The samples were washed and pre-incubated for 30 minutes at RT with 2% BSA in PBS to block non-specific binding sites. After the washing step, the sections were incubated with primary antibody diluted 1:50 in 0.1% BSA in PBS overnight at 4°C. After washing with PBS three times, secondary Alexa Fluor 488 antibodies (diluted 1:200 in 0.1% BSA in PBS, goat Anti-Rat-IgM, Thermo Fisher Scientific, Denmark) were added and the sections were incubated overnight at 4°C. Then, the incubated sections were washed in PBS and MiliQ water and finally counterstained with Calcofluor White (Sigma Aldrich, USA). An Olympus BX51 CLSM microscope with corresponding software FluoView v.5.0. (Olympus Corporation, Tokyo, Japan) was used for imaging. In order to perform control reactions, the incubation with the primary antibody was omitted. All photographs, figures, and schemes were edited using the CorelDrawX6 graphics program.