Lsm 710 confocal workstation

Fresh eyes were enucleated and fixed in 2% paraformaldehyde (PFA) for 10 min and then the anterior parts including cornea, lens, and attached iris pigmented epithelium were removed. The resulting posterior eyecups were fixed in 2% PFA for 1 h at room temperature either for cryosections or RPE flat mount. For cryosections, the eyecups were dehydrated through gradient sucrose solutions and embedded in OCT. For RPE flat mounts, retinas were then removed after the eyecup was quartered like a petaloid structure. The resulting eyecup was further cut radially into eight pieces from the optic nerve head to the periphery. Immunostaining on flat mount and cryosections was performed as described previously (Zigler et al., 2011). Stained RPE‐choroid sheets with sclera were mounted on a microscope slide with RPE layer up. Images were acquired by Zeiss LSM 710 confocal workstation.

The retrovirus-based pLNCX2 vector (Clontech) derived from MMLV was used to obtain stable expression of mRFP in cultured B16-F10 and WM793B cells (as per manufacturer’s protocol). Cultures of MSCs infected with vMyx-EGFP (MOI = 10) were trypsinized and added to mRFP-expressing B16-F10 or WM793B cultures (1:2 cell-to-cell ratio). Infection progress was evaluated using fluorescence microscopy (Zeiss LSM 710 confocal workstation).

Samples of bone marrow collected from healthy donors (MSC Memorial Cancer Center, Gliwice, Poland) were diluted with Minimal Eagle’s Medium (MEM) supplemented with 10% FCS (Eurex), 1% antibiotics (Sigma), and 1% non-essential amino acids (Sigma) and transferred to a humidified incubator (37°C, 5% CO₂). After 48–72 h, cultures were washed with PBS⁻ (without Mg²⁺ and Ca²⁺ ions) to remove non-adherent cells. Sub-confluent cultures were passaged at a 1:3 split ratio. Cells from passages 2–4 were used for further experiments. Differentiation into osteocytes and adipocytes was analyzed at passage 2, using the Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems, SC006) containing goat anti-mouse FABP4 antigen affinity-purified polyclonal antibody (adipocyte marker) and mouse anti-human osteocalcin monoclonal antibody (osteocyte marker). Cells were stained with Biotinylated Rabbit Anti-Goat IgG (immunoglobulin G) Antibody (H+L) and Texas Red Streptavidin (Vector Laboratories, BA-5000 and SA-5006, respectively) or Goat Anti-Mouse Alexa Fluor Plus 488 secondary antibody (Thermo Fisher Scientific, no. A32723). Nuclei were counterstained with DAPI (Thermo Fisher Scientific, no. 62248). Morphology of bone-marrow-derived cells was inspected using the Zeiss LSM 710 confocal workstation.