Dmrxa microscope

Heads were collected from euthanized mice and fixed in 4% buffered paraformaldehyde (PFA) in phosphate buffered saline 0.1 M (PBS) for 48 h. Heads were decalcified in 4.13% EDTA/0.2% PFA pH 7.4 in PBS for 4 days in a KOS sw10 (Milestone, Sorisole, Italy). The samples were dehydrated and embedded in paraffin or maintained in a PBS buffer solution at 4°C before cryostat sectioning. Then, 3-μm-thick frontal sections stained with Hematoxylin-eosin and Masson's trichrome (three-color staining protocol, which stains muscle fibers in red, collagen and bone in green, cytoplasm in light red, and nuclei in dark brown) were observed using a DMRXA microscope (Leica, Nussloch, Germany). Tartrate resistant acid phosphatase (TRAP) staining was performed as previously described (Castaneda et al., 2011 (link)) to identify the multinucleated osteoclast cells after a 90 min incubation in a 1 mg/mL Naphtol AS-TR phosphate, 60 mmol/L N,Ndimethylformamide, 100 mmol/L sodium tartrate, and 1 mg/mL Fast red TR salt solution (all from Sigma Chemical Co., St Louis, MO, USA) and counterstained with hematoxylin.

The heads were collected from the euthanized mice and fixed in 4% buffered paraformaldehyde (PFA) in phosphate buffered saline 0.1M (PBS) for 48 hours. The heads were decalcified in 4.13% EDTA/0.2% PFA pH 7.4 in PBS for four days in a KOS sw10 (Milestone, Sorisole, Italy). The samples were dehydrated and embedded in paraffin or maintained in a PBS buffer solution at 4°C before cryostat sectioning. Then, 5µm-thick frontal sections stained with Masson’s trichrome were observed using a DMRXA microscope (Leica, Nussloch, Germany).
Tartrate-resistant acid phosphatase staining (TRAP, Sigma Chemical Co., St Louis, MO, USA) was performed as previously described [8 (link)] to identify the multinucleated osteoclast cells.

For immunofluorescence, HIEC-6 cells were fixed in MeOH (Sigma-Aldrich, St. Louis, MO, USA) for 10 min at −20 °C, and nonspecific sites were blocked for 1 h at room temperature with 10% Blotto-PBS (pH 7.4). Primary antibodies were diluted 1:1000 in a blocking solution containing 0.05% azide and incubated overnight at 4 °C. The secondary antibodies were used at 1:400 dilution in the blocking solution and incubated at room temperature for 1 h. Nuclei were stained with DAPI 1:3000 (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 3 min at room temperature. The slides were visualized with a DMRXA microscope (Leica, Nussloch, Germany) equipped for epifluorescence and digital imaging (RTE/CCD Y/Hz-I300 cooled camera). Images were acquired using MetaMorph software (Universal Imaging Corporation, New York, NY, USA) with 20× and 40× objectives. Fluorescence was quantified with ImageJ™ software, JACoP plugin (NIH, Bethesda, MD, USA).

For analysing the sporulation of FtsZ*strains, cover glasses were positioned in SFM plates under an angle of 45°, and subsequently, 10 μl of a spore suspension were inoculated (10⁸ spores/ml). After 5–7 days, depending on the strain, cover glasses were removed, mounted with ultrapure mQ water, and observed using phase contrast under the Leica DMRXA microscope. Pictures were taken with an ORCA-Flash4.0 V3 Digital CMOS camera.

5 million cells were harvested 24 hr after drugs were washed out for metaphase analysis as described (Callén et al., 2007 (link)). Quantitative FISH analysis using a Cy3-labeled (CCCTAA) peptide nucleic acid probe (Applied Biosystems) was performed as described previously (Callén et al., 2007 (link)). Telomere length measurements were performed on least 15 metaphases for each cell type. DAPI chromosome and Cy3 telomere images were acquired with a constant exposure time that ensured all captured fluorescent signals were within the linear range using Metafer Software on a Zeiss Axios Imager Z2 Microscope. Image analysis was done in the Metafer ISIS software. SKY was performed and analyzed as previously described, using the hiSKY 7.2.7 Software (ASI) on a Leica DMRXA Microscope (Liyanage et al., 1996 (link)). A minimum of 35 metaphases were imaged and analyzed.

Certain macroscopically homogeneous samples were subjected to optical microscopy using a DMR-XA microscope from Leica (Wetzlar, Germany). The modes of observation included halogen illumination and polarized light over a dark field. Then, the particle size was analyzed using the open-source software ImageJ (Version 1.53), as described elsewhere [6 (link)], but without using the Fractal Box Count plugin. Instead, the average between the maximum Feret’s diameter and the minimum Feret’s diameter, readily available in the native software package, was taken to indicate the particle size (d). OriginLab’s OriginPro 8.5 was then used to fit the resulting size distributions.
The viscosity of the stable emulsions, including different degrees of oxidation and concentrations of TOCNFs, was measured using a rheometer with concentric cylinder geometry from PCI (Albacete, Spain), model RVI-2. The radii of the spindle and the outer cylinder were 4 mm and 5 mm, respectively. The rheological behavior of the TOCNF-stabilized BW-in-water emulsions was compared with that of the aqueous TOCNF suspensions at the same consistency. The effects of temperature and the influence of the shear rate were studied.