Axioimager m2 imaging microscope

Senescence-associated β-galactosidase (SA-β-gal) staining is the gold standard method for quantifying cellular senescence (50 (link)), and has recently been shown to sensitively measure changes in the spinal cord following injury in the mouse (28 (link)). Lumbar L4/5 spinal cord samples were isolated, postfixed in 4% paraformaldehyde for 2 hours, cryoprotected, and embedded in OCT medium. Frozen spinal cord sections (20 μm thick) were cut using a cryostat (Leica) and mounted onto Superfrost Plus slides. The Senescence β‑Galactosidase Staining Kit (Cell Signaling Technology, catalog 9860) was used to visualize the senescent cells, per the manufacturer’s instructions. Briefly, the lumbar spinal cord tissue sections were washed with 1× PBS (pH 7.4; 2 × 10 minutes) and were incubated with the SA‑β‑gal staining solution (pH 6.0) in the dark at 37°C (dry incubator; no CO₂) for 24 hours. After the incubation period, the sections were washed with 1× PBS (3 × 10 minutes), coverslipped, and visualized using a Zeiss AxioImager M2 imaging microscope. Experimenters were blinded to the surgery group and sex of the mice during quantification.

For p53 colocalization with different cell types, tissue sections from all experimental groups were imaged by Zeiss AxioImager M2 Imaging microscope with Zeiss ZenPro software v2.3 (Zeiss Canada). Acquisition settings were held constant for all images being compared by quantitative analysis. For representative images, Z-stacks were taken using a ×63 objective on a Zeiss LSM 880 confocal microscope. Three-dimensional and surface-rendered illustrations were prepared by Imaris software. Experimenters were blinded to the surgery group and sex of the mice during quantification.