Bx50 microscope

For the analysis of bone volume, MMA-embedded sections were stained with von Kossa/van Gieson staining. To identify osteoblasts and osteoclasts, bone sections were stained using toluidine blue and tartrate-resistant acid phosphatase (TRAP) staining, respectively. Bone cell number/mm trabecular bone surface as well as dynamic histomorphometric parameters were analyzed using the OsteoMeasure software attached to an Olympus BX50 microscope, × 20 objective (Olympus UPlan Fl 20x/0.50 ∞/0.17) according to the standards of the American Society for Bone and Mineral Research (ASBMR) [37 (link)]. The tumor volume and the trabecular bone volume in the femora of mice were quantified using hematoxylin and eosin- or van Giemsa-stained sections (2 non-serial sections per mouse, 20 μm apart) 9 days after tumor cell injection. The analysis was performed using the OsteoMeasure software and an Olympus BX50 microscope (× 10 objective, Olympus UPlan Fl 10x/0.30 ∞/-). The total tumor area and the trabecular bone area (Additional file 1: Figure S1B) of a tissue area with a length of 2700 μm were quantified.

Alternate sections were deparaffinized, rehydrated and stained with haematoxylin-eosin (Bio Optica, Milan, Italy) according to the standard procedure. The sections were then observed using a light optical microscope (Olympus BX50 microscope, Hamburg, Germany). According to Guidotti et al. [44 (link)], a blind examiner identified MD, BD and MT hepatocytes using the Olympus BX50 microscope at a final magnification of 1000×. The number of all polyploid hepatocytes was evaluated in 10 random fields in each liver sample.

The stomatal pore condition and xylem structure were analyzed using an Olympus BX50 microscope (Olympus, Tokyo, Japan). The preparation of leaf samples for stomatal analysis was described previously in [18 (link)]. Fresh leaflets were collected from plants under control and water-withholding stress conditions, and a thick tape was pasted on their upper surface. The tape was gently pulled from the leaflet to tear off the epidermis layer and placed on a glass slide. Other leaf parts were cut off using a scalpel. A coverslip was placed on the sample after adding a drop of water. The slides were observed at 1000× magnification to determine the stomatal pore area. Due to their elliptical shape, the following formula was used to calculate the stomatal pore area: $$\mathrm{Stomatal} \mathrm{pore} \mathrm{area} \left(\mathsf{\mu }\mathrm{m}²\right)=\mathsf{\pi }\times r_1\times r_2$$
where π = 3.14, and r₁ and r₂ are the minor and major radii of the stomatal pores, respectively.
To observe the xylem structure, a cross section of the stem was obtained from one-month-old plants under control and drought stress using a scalpel. Then, the sections were immersed in 0.05% toluidine-blue-O (TBO) (Waldeck, Münster, German) for 30 s and washed with dH₂O several times. The sections were then examined under 100× A magnification using an Olympus BX50 microscope (Olympus, Tokyo, Japan).