Bx51 upright microscope

A metabolically active cell density at day 0 and day 7 was determined using the MTT assay and 4′,6‐diamidino‐2‐phenylindole (DAPI) (both Sigma–Aldrich, Ireland) counterstaining as previously described.⁶, ⁷¹, ⁷² Freshly isolated and cultured discs were cut in half along the sagittal plane and an approximate 3 mm thick section was taken across the full diameter of the disc. The sections were incubated in high‐osmolarity phenol‐free DMEM with 0.5 mg/mL MTT for 3 hours under gentle agitation at 37°C. Tissue was washed, embedded in optimal cutting temperature compound and stored at −80°C until sectioning on a cryotome. For cell density analysis, 10 μm slices were cut, randomly selected for mounting on a microscope slide using fluoroshield with DAPI. Slides were examined under both brightfield, to capture active MTT+ cells, and fluorescence, to capture all cell nuclei stained with DAPI (Olympus BX51 upright microscope). The metabolically active proportion was assessed qualitatively by merging the brightfield and DAPI image using ImageJ (National Institutes of Health, and Bethesda, Maryland). Images for the NP and AF regions at day 0 and day 7 can be found in Supplementary Figure S1. The total number of cells (DAPI) was determined automatically while the number of dual stained cells (overlapping formazan crystals and blue nuclei) was performed manually using the cell counter plug‐in.

Cells expressing BirA* constructs were plated onto glass coverslips for 24 h and incubated with 50 µM biotin for 24 h to initiate biotinylation of proximal proteins. Cells were fixed with 4% (wt/vol) PFA for 15 min at RT and permeabilized with 300 µl of 0.2% (wt/vol) Triton X-100 for 20 min at RT. Coverslips were incubated with primary antibodies directed against proteins indicated in 2% (wt/vol) BSA in PBS for 1 h at RT. Cells were then incubated with fluorophore-conjugated secondary antibodies at RT for 20 min and stained with 1 µg/ml DAPI for 1 min before washing and mounting onto glass slides. Images were acquired using an Olympus BX51 upright microscope with a 60×/0.65-1.25 UPlanFLN or 10×/0.30 UPlanFLN objective and captured using a Coolsnap EZ camera (Photometrics) through MetaVue software (Molecular Devices).