Ha binding protein

In order to further study the role of HA on MG development, the 3D hanging drop culture system was used. Ten-microliter drops of hMGCs at a density of 2.5 × 10⁴ cells/mL were placed on the inside of a 60-mm petri dish lid (Eppendorf, Hauppauge, NY, USA). Various brands were tested, and the Eppendorf-treated cell culture dishes presented the highest surface tension and were, therefore, the most suitable for the hanging drop technique. The hMGCs were placed in culture in the presence or absence of HA, specifically 0.01% ULMWHA, 0.05% LMWHA and 0.05% HMWHA were used. The lids were then inverted and placed over a petri dish containing 1 mL PBS to prevent the drops from drying out. On day 4, FBS at a final concentration of 10% was added to the cells to trigger differentiation, and the cultures were maintained for a further 6 days. The cells were maintained at 37°C and 5% CO₂ for a total of 10 days after which images were taken and the spheres were fixed in 2% paraformaldehyde for 30 minutes. The spheres were washed with PBS, blocked, and stained with Alexa Fluor 488 Phalloidin (Cell Signaling, Eugene, OR, USA) and HA binding protein (Millipore) followed by NeutrAvidin Alexa Fluor 555 conjugate. Nuclei were counterstained with DAPI.

Eyelids were collected and immediately fixed in 4% buffered paraformaldehyde overnight and then embedded in Tissue-Tek embedding medium (Sakura Finetek USA, Inc., Torrance, CA, USA) for cryosectioning. Sections (10 μm) were cut using a Leica CM 1950 (Leica, Buffalo Grove, IL, USA) cryostat and collected on Fisherbrand SuperfrostPlus Gold microscope slides (Thermo Fisher Scientific). Upon use, sections were incubated for 30 minutes at 60°C, and excess tissue embedding medium was removed with PBS. Unspecific protein binding sites were blocked with 10% fetal bovine serum (FBS) prepared in PBS containing 0.01 M saponin. Sections were then incubated with the primary antibodies anti-Krt14 (PRB-155P; Covance, Princeton, NJ, USA) and HA binding protein (Millipore). Sections were washed and incubated with NeutrAvidin Alexa Fluor 555 conjugate and anti-rabbit produced in donkey conjugated with Alexa 488 for 1 hour at room temperature. A secondary control was carried out with rabbit IgG isotype control (Abcam, Cambridge MA, USA) in place of the primary antibody and did not yield any staining (results not shown). Slides were mounted in Fluoromount-G and imaged under an LSM 800 confocal microscope (Zeiss).