Szh10 microscope

Right lungs were inflated using formaldehyde and fixed for 24–48 hours before being moved to 70% ethanol. The lungs were sectioned and subjected to Masson’s trichrome staining and immunostaining for smooth muscle α-actin (α-SMA) or fibronectin. Secondary antibodies conjugated to either HRP (immunohistochemical images) or Alexafluor-488 (for quantitation of α-SMA) were utilized. The modified Ashcroft scale was used to quantify fibrosis, as previously described [17 (link)]. Quantitation of α-SMA immunostaining was performed by taking 10 images at 20X magnification per sample, using identical exposure times and thresholds on an Olympus 1X71 fluorescent microscope and Q imaging Retiga 2000R camera. ImageJ was used to measure integrated density of the fluorescent signal [18 (link)]. Quantitation of fibronectin histology was performed by taking a 2X magnification image of each sample, using identical exposure times and thresholds on an Olympus SZH10 microscope and Olympus DP70 camera. ImageJ Immunohistochemistry Toolbox plugin was utilized to isolate the brown stain [19 ]. The resulting image was inverted, the lung was outlined and integrated density measured. Integrated density measurement was divided by the total area of the lung in order to account for differences in lung sizes.

In the exercised and control subjects, bone formation on the tibial periosteal surface during the treatment period was quantified on midshaft transverse cross sections (100-µm thickness) using an Olympus SZH-10 microscope (Olympus America, Melville, NY) with epifluorescence (Fig. 1). Using ImageJ, digitized cross sections were divided into 16 equal-angle sectors positioned about the experimentally determined neutral axis, with the axis orthogonal to the neutral axis projected through the area centroid [8] (link). Bone area added in each sector during the treatment period was measured from the calcein line to the periosteal surface. Bone areas were standardized by body mass^0.67, calculated as mean mass during the final 3 weeks of the experiment. Two-sample Wilcoxon tests were performed to evaluate differences in bone formation between exercised and control animals.
Additional details of the experimental and analytical methods are provided elsewhere [8] (link), [11] (link). Raw bone strain and histological data are available in [8] (link) and Dataset S1, respectively.

For CFU assays, cells were seeded in limiting dilution, i.e., 2 cells/cm², incubated for 14 days, fixed with 4% formaldehyde, and stained with 0.1% Azur II (Sigma Aldrich, Taufkirchen, Germany) dissolved in Aq_dest for 20 min at room temperature (RT), air dried, and photographed with a SZH10 microscope (Olympus, Münster, Germany) equipped with a CCD Colour view III camera (Olympus, Münster, Germany). The resulting images were taken using the Cell Olympus cell Sens software version 1.5 (Olympus, Münster, Germany). Images were analyzed using the ImageJ plugin “analyze particles” following threshold adjustment. Stained colonies of ≥50 cell were scored as colony forming (CFU-F) and counted. CFU-F efficiency was calculated as follows:

T. pretiosum adults were preserved in 3% glutaraldehyde in 0.025 M pH 7.0 phosphate buffer for 24 h and then used for fixation with a Pelco Biowave processor (Ted Pella Inc., Redding, CA, USA). A critical point drying apparatus (Polaron E3000, Quorum Technologies, Lewes, UK) was used in the preparation process at the critical point of CO₂ = 31.1 °C and 1071 psi. Dried samples were mounted on SEM stubs under a SZH10 microscope (Olympus Corporation, Tokyo, Japan) and then sputtered with 10 nm gold using a Polaron Sputter coater SC 7640 (Quorum Technologies, Lewes, UK) with argon gas (pressure < 1 × 10⁻² mbar, voltage = 1 kV). Samples were examined and photographed under a Zeiss 1555 VP-FESEM SEM instrument (ZEISS Australia, North Ryde, Australia) operated at 10 kV, high current, 10–12 mm working distance, and 30 µm aperture. Sample preparation and examination were conducted at the Centre for Microscopy, Characterization, and Analysis (CMCA) at the University of Western Australia. Five male and five female antennae were observed under SEM (n = 5).

The viability test with 2,3,5-triphenyltetrazolium chloride (TTC) was used to obtain insights into metabolic processes. The TTC metabolic assay exploits enzymatic processes that occur in all living cells and organisms, such as oxidation-reduction reactions during photosynthesis or respiration. In this assay, the clusters of ESEs were incubated in a colourless 1% (w/w) solution of 2,3,5-triphenyltetrazolium chloride (TTC) for 24 hours. The TTC is taken up by the live cells and reduced to a water-insoluble red formazan by oxidative enzymes. Dead cells remain colourless after incubation with TTC. After 24 hours of incubation at room temperature, the results were photographed using an Olympus (SZH 10) microscope connected to an Olympus (4040 Zoom) digital camera.