Alcian blue 8gx

Chondrogenic cell pellets were collected at day 21, fixed with 10% formalin, and embedded in paraffin for histology sectioning. Embedded specimens were sectioned at a thickness of 7 µm. For hematoxylin and eosin (H&E) and Alcian blue staining, slides were prepared following our laboratory standard protocol before being stained with Gill’s Hematoxylin #3 and 0.5 % Eosin Y or Alcian blue 8GX (Polysciences, Warrington, PA, USA). Immunohistochemical analysis was performed by blocking rehydrated samples with 1 % BSA in PBS and then incubating the samples with mouse anti-human collagen type 2 primary antibody (Millipore, Billerica, MA, USA) diluted in PBS at 1:200 for 1 h. Samples were then washed with PBS and incubated with FITC-conjugated secondary antibody (eBioscience, San Diego, CA, USA) diluted 1:100 in PBS for 1 h. Coverslips were affixed to slides using Prolong antifade reagent with DAPI (Life Technologies) prior to imaging.

Chondrogenic cell pellets were collected at day 21, fixed with 10% formalin, embedded in paraffin, and sectioned into 7 μm-thick slices. Slides were prepared following our laboratory standard protocol prior to staining with Gill’s Hematoxylin #3 and 0.5% Eosin Y or Alcian blue 8GX (Polysciences). Immunohistochemical analysis was performed by blocking rehydrated samples with 1% BSA in PBS and then incubating the samples with mouse anti-human collagen type 2 primary antibody (Millipore) diluted in PBS at 1:200 for 1 hour. Samples were then washed with PBS and incubated with FITC-conjugated secondary antibody (eBioscience) diluted 1:100 in PBS for 1 hour. Coverslips were affixed to slides using Prolong Antifade Reagent with DAPI (Life Technologies) prior to imaging.