Camedia c4040

3 × 10⁴ cells of each of the cell lines were seeded on coverslips for 24 h in their respective media. Then, cells were fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.2% Triton X-100 in PBS for 20 min. Cells were blocked with blocking buffer (10% goat serum, 1% BSA, 0.2% triton X-100, and PBS 1X) for 1 h and then stained with the primary antibodies: mouse monoclonal anti-E-Cadherin antibody (Clone: 36/E-cadherin; BD Biosciences, San José, CA, USA), rabbit monoclonal anti-Vimentin antibody-Alexa Fluor-594 (Clone: EPR3776; Abcam, Cambridge, MA, USA), overnight at 4°C. After that, cells were incubated with the secondary antibody goat anti-mouse-IgG-FITC antibody (Sigma-Aldrich Co., St. Louis, MO, USA) for 30 min. Nuclei were stained with DAPI for 25 min. Cells were observed using a fluorescence microscope Olympus BX51, and images were acquired with a digital camera (Camedia C4040, Olympus).

Leaf anatomy was measured on the fully expanded second leaves from five pepper seedlings at a similar position for each replication each treatment [46 ] on 28 DAT. Leaf segments of 5 mm × 5 mm were taken from the central leaf blade next to the main vein, fixed with formalin-acetic acid-alcohol (FAA) fixative, dehydrated in an alcohol and xylene series, embedded in paraffin, cross-sectioned to a thickness of 10 μm, and stained with red-solid green. The total thickness of the whole leaf and the thickness of the upper epidermis, lower epidermis, PT and SPT were measured under a transmission light microscope (DP71, Olympus Inc., Tokyo, Japan). Images were collected using a digital camera (Camedia C4040, Olympus Inc., Tokyo, Japan) and analyzed by AnalySIS 5.0 (Olympus Inc., Tokyo, Japan).

Undecalcified sections of 30 intact samples from each group were prepared. The histomorphometric examination was performed as described by our previous study [16 (link)]. The specimens were fixed in 10% buffered formalin, dehydrated in increasing concentrations of ethanol of 70% to 99% for 10 days, and embedded in methylmethacrylate (Technovit 7200VLC; Heraeus Kulzer GmbH, Wehrheim, Germany). The 50 μm thick sagittal sections were prepared using an electric diamond saw and grinding system (Exakt; Exakt Vertriebs, Norderstedt, Germany) and stained with toluidine blue. Digital images of the sections were obtained using a digital camera (Camedia C4040; Olympus, Tokyo, Japan) attached to an Olympus BX50 microscope (Olympus) at a magnification rate of 2x (Figures 4 and 5). The images were transferred to a personal computer, and a standardized measurement area (2 × 2 mm) was outlined in the middle of the condylar bone. Bone volume, trabecular width, trabecular thickness, trabecular separation, and node/terminus ratio measurements were made using histomorphometry software (TAS, version 1.2.9; Steve Paxton, University of Leeds, Leeds, West Yorkshire, UK). The nomenclature and calculations for bone histomorphometry were applied in accordance with the report from the American Society for Bone and Mineral Research [18 (link)].

At harvest, mature leaves were sampled from five plants per treatment, fixed in a 38% formaldehyde, glacial acetic acid, 50% ethanol solution (5:5:90 by volume) and prepared for microscopy according to standard protocols (De Micco et al., 2011 (link); Carillo et al., 2020 (link)). In brief, subsamples of leaves were dehydrated and embedded in the acrylic resin JB4 (Polysciences, Hirschberg, Germany) and thin sectioned (5-μm thick) through a rotary microtome. The cross sections, stained with 0.5% Toluidine blue in water, were observed through a transmitted light microscope (BX60; Olympus, Hamburg, Germany). Digital images were captured (Camedia C4040; Olympus, Hamburg, Germany) and subjected to digital image analysis (AnalySIS 3.2; Olympus, Hamburg, Germany) to quantify the following traits: the thickness of leaf lamina, palisade, and spongy parenchyma, the quantity of intercellular spaces in the palisade parenchyma (in six regions per section). Another set of subsamples of leaves was devoted to peeling, and microphotographs of epidermal strips of the upper and the lower lamina surfaces were analyzed to quantify the following stomata traits: stomata frequency (number of stomata per mm² in three regions per peel) and stomata size (length and width of the guard cells in 10 cells per lower epidermis peel).