Fluoview fv10i

F- and G-actin were visualized from Legionella-infected macrophages using Alexa Fluor® 568 phalloidin and Alexa Fluor® 488 DNAse I (Molecular Probes) and for studies examining colocalization of GFP-expressing bacteria (Legionella and E. coli) with the lysosome, Lysotracker™ red was used to stain acidic vesicles of infected BMDMs, as previously described14 (link). To examine cell death, after infection macrophages were stained with 4 uM ethidium homodimer-1 (EthD-1) from the LIVE/DEAD^® viability/cytotoxicity kit (Molecular Probes), according to manufacturer’s instructions. Images were captured using laser scanning confocal fluorescence microscope with a 60X objective (Olympus Fluoview FV10i).

Transverse frozen sections were cut from the mid-belly of the TA or diaphragm muscle strips at a thickness of 8 μm, mounted on slides and stored at −80 °C until analysis. Immunohistochemistry for ADAMTS1 (Origene, TA317919, Rockville, MD, USA), ADAMTS5, ADAMTS15 (Abcam, ab45047, Cambridge, MA, USA), V0/V1 versican (anti-GAGβ; Merck Millipore, AB1033, Bayswater, VIC, Australia) or versikine (anti-DPEAAE neo-epitope; Thermo Fisher Scientific, PA1-1748A, Scoresby, VIC, Australia) were performed as previously described [9 (link),32 (link)]. An anti-desmin rabbit polyclonal antibody (Abcam, ab15200, Cambridge, MA, USA) together with an anti-rabbit Alexa Fluor 488 secondary antibody (Thermo Fisher Scientific, R37116,) were used to detect myoblasts and newly regenerated myofibres [93 (link),94 (link)]. Representative wild type and mdx TA and diaphragm muscle cross-sections were H and E stained for muscle architecture, and wheat germ agglutinin to assess fibrosis [95 (link)]. For analysis of V0/V1 versican and versikine immunoreactivity, four non-overlapping representative digital images were captured with a confocal microscope of each muscle section at 600× magnification (Olympus Fluoview FV10i) and analysed for area of immunoreactivity using Image-Pro Plus software (Version 7, Media Cybernetics, Silver Spring, MD, USA).

The Live and Dead assay stain solution was a mixture of two highly fluorescent dyes that differentially labelled live and dead cells. The Live and Dead cell assay was a one-step staining procedure. It could be used directly in cell culture media.
For qualitative and quantitative analyses, all ARPE-19 cell samples were stained with the fluorescent dye Live and Dead (ab 115347 Abcam, Cambridge, UK) according to the protocol presented by the manufacturer. For the qualitative analysis, the cell culture was washed three times with phosphate buffer solution (PanEco, Moscow, Russian) and fixed in 10% formalin (PanEco, Moscow, Russian) for 20 min. Incorporation of LGs was monitored with a confocal microscope (Olympus Fluoview FV-10i, Tokyo, Japan) using the green channel AF 488 (Ex 490 nm/Em 525 nm) and red channel AF 594 (Ex 590 nm/Em 617 nm).
For the quantitative analysis, the RPE cell suspension was obtained with the enzymatic removal of cells from the culture plastic, washed three times in CellWash solution (BD, Franklin Lakes, NJ, USA), and analyzed with a flow cytofluorometer (CytoFlex, Beckman Coulter, Brea, CA, USA) using the detection channels FITC (Ex 493 nm/Em 528 nm) and Cy3 (Ex 550 nm/Em 615 nm).

Matured BC-OCs were seeded on cortical bone slices (0.2 mm thick) and allowed to resorb for 3 days as described in the section Bone resorption assays and analyses. Cells were fixed with 3.7% formaldehyde for 15 min, washed with PBS, and stained with fluorescently labeled phalloidin for 20 min. Bone slices were washed in PBS, mounted in ProLong Gold containing 4′,6-diamidino-2-phenylindole (Invitrogen), covered with a coverslip, and sealed with clear nail polish. Confocal images were obtained using an Olympus Fluoview FV10i (Olympus Corporation, Shinjuku, Tokyo, Japan) and images were processed using Imaris version 7.6.5 (Bitplan AG, Zurich, Switzerland).

To verify the identity and assess the purity of isolated glial cells, immunofluorescent microscopy was used. Cells plated on poly-D-lysine-coated coverslips were fixed in acetic acid/ethanol [5:95 (v/v)] at −20 °C for 10 min and then incubated with the relevant primary antibody for 30 min at room temperature, followed by three washes in Minimum Essential Medium (MEM) (Invitrogen) and subsequent incubation with secondary antibody for 30 min. Sections were mounted in a medium containing 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI) (100 ng/mL; Vector Laboratories) to show cell nuclei, and examined by confocal microscopy (Olympus FluoView FV10i; Olympus, Tokyo, Japan). The primary antibodies used were anti-GFAP (rabbit polyclonal antibody; 1:1000; Cat#ab7260, Abcam, MA) for astrocytes, anti-GalC (mouse monoclonal antibody; 1:100; Cat#MAB342, Merck Millipore) for oligodendrocytes, and anti-A2B5 (mouse monoclonal antibody; 1:100; Cat#MAB312, Merck Millipore) for OPCs. The secondary antibodies used were Dylight 488-conjugated goat anti-rabbit IgG (1:200; Cat#E032220-2, EarthOx) and Alexa Fluor 594-conjugated goat anti-mouse IgG (1:200; Cat#A-11005, Invitrogen).

Cell viability was evaluated using a Molecular Probe Live-Dead cell imaging system (Invitrogen). Human brain organoids were harvested for cell viability analysis at days 4, 5, 7, 10 and 21. Organoids were incubated at room temperature for 10 minutes in DPBS containing 2 μM calcein AM (Invitrogen, green- live cells) and 4 μM ethidium homodimer-1 (Invitrogen, red - dead cells). After washing once with DPBS, the organoids were then imaged using the Olympus Fluoview Fv10i (Olympus, Tokyo, Japan) laser scanning confocal microscope. The images obtained were then quantified using ImageJ to determine cell viability percentages.

Cells were seeded in Lab-Tek II Chamber (Nalge Nunc International, Rochester, NY, USA), fixed with 3.7% methanol-free formaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 5 min and incubated with 3% BSA in PBS for 45 min. Slides were stained with ALDH1L1-specific in-house rabbit antibody (1: 1000) and PSMA6-specific (proteasome 20S alpha 6 subunits) mouse monoclonal antibody (OriGene, cat# TA800104; 1:100) at 4 °C overnight, washed three times with PBS and then incubated with secondary chicken anti-rabbit antibody conjugated with Alexa Fluor 488 (Thermo Fisher Scientific, cat. # A-21441; 1: 200) and goat anti-mouse IgG2a secondary antibody conjugated with Alexa Fluor 555 (Thermo Fisher Scientific, cat. # A21137; 1: 200) in dark chamber at room temperature for 1 h. Slides were washed twice with PBS and mounted with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Images were captured using confocal laser scanning microscope Olympus FluoView FV10i (Olympus, Tokyo, Japan).