p16INK4a staining was performed for both monolayer cultures and pellet samples. Only the pellet samples were heated on an iron heater at 50 °C for 30 min and rehydrated by PBS-T (0.1% Triton X-100) for 10 min. Both healthy monolayer cultures and pellet samples were blocked with hydrogen peroxide for 10 min, washed three times, and saturated with 1% BSA, 1% goat serum, and 0.1% Triton X-100 for 10 min. All samples were incubated at 4 °C overnight for p16INK4a antibody (CINTec Kit, Roche) and PBS-T for negative control. The HRP/DAB Detection IHC Kit (Abcam, ab64264) was used for detection. Counting staining was applied with Meyer’s hematoxylin (Sigma-Aldrich, Oakville, ON, Canada) for 2 min. Samples were rinsed with water (30 s), 75% ethanol (15 s), and 95% ethanol (15 s) afterwards and coverslips were mounted with Permount™ Mounting Medium (Fisher Scientific). Images were captured as described [18] for Safranin-O staining, and analyzed with Fiji Image J (version 2.1.0/1.53c).

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