Pannoramic scan digital slide scanner

Samples were fixed in 4% paraformaldehyde (24 h, 4 °C), dehydrated, and embedded in paraffin, according to standard histological protocols. Sections (5 μm) of tissues were mounted on SuperFrost Plus slides (Thermo Fisher, Waltham, MA, USA). After dewaxing and rehydratation, sections were heated at 97 °C in 10 mM citrate buffer (pH 6.0) for 40 min. Nonspecific binding was blocked using protein block serum-free (X0909; DakoCytomation) for 1 h at room temperature. Sections were incubated with primary antibodies diluted in antibody diluent (S3022; DakoCytomation) overnight at 4 °C. The primary antibodies used were anti-Z0–1 (1:500; LifeTech), anti-claudin-1 (1:500; InVitrogen), anti-claudin-2 (1:500; InVitrogen), anti-PCNA (1:1000; GeneTex) and anti-Ki67 (1:50, DakoCytomation). Nuclei were stained with Hoechst before mounting the slides using Fluorescent Mounting Media (Dako). Sections were scanned using a Pannoramic scan digital slide scanner (3DHistech) and analysed using digital slide scanner Pannoramic scan (3Dhistech). For Ki67 and PCNA 10 crypts were measured per mice and per cut.

Plants destined for histology were grown for 3 weeks in short day (SD) conditions (8 h light and 16 h dark) and then transferred to long day (LD) conditions (16 h light and 8 h dark) to induce flowering and used for histology at a height of 15–20 cm. Stem segments of at least 1 cm in length (including the stem base) were harvested, embedded in paraffin and sectioned using a microtome (10 µm sections). After deparaffinization, sections were stained with 0.05% toluidine blue (Applichem), fixed with Entellan (Merck) and imaged using a Pannoramic SCAN digital slide scanner (3DHistech). Pictures were analysed in a blind test using the Pannoramic Viewer 1.15.4 software (3DHistech). For quantitative analyses, at least five plants were analysed for each data point.