Hematoxylin counterstain

Livers were sectioned, formalin-fixed paraffin-embedded (FFPE) and stained with hematoxylin-eosin (H&E) for assessing NAFLD activity score (NAS), as described. 13 (link) To assess fibrosis, deparaffinized and rehydrated slides were stained with 0.1% Sirius Red stain (Direct Red 80, Sigma-Aldrich, St Louis, MO) and imaged by Nikon Eclipse 90i Microscope (Nikon, Melville, NY), as described. 9 (link) For immunohistochemical analysis, FFPE sections were deparaffinized in xylene, progressively rehydrated in alcohol gradient before quenching peroxidases with 3% H 2 O 2 and . heat-induced antigen-retrieval using 10mM sodium citrate buffer (pH 6.0) or Tris-buffer (pH 10.0), per antibody requirement. The Abcam antibodies [α-CD68 (1:150), Mac2 (1:250), MPO (1:1000), CD4 (1:1000), CD8 (1:2000), and α-Ki67 (1:100, monoclonal)] and α-Cleaved Caspase 3 (1:200, Cell Signaling, Danvers, MA) were used. This was followed by blotting with ImmPRESS HRP horse anti-mouse or anti-rabbit (Vector labs, Newark, CA) for 30min before being treated with 3,3'-Diaminobenzidine (DAB, Vector labs) and hematoxylin counterstain (Vector labs). Sections were evaluated blindly and positively stained cells were counted in 5 fields/mouse at 40X magnification.

Mouse hindlimbs were fixed in formalin, decalcified in formic acid, embedded in paraffin and sectioned for immunohistochemistry. Sections were incubated overnight at 4 C with antibodies against acetylated a-tubulin (Sigma), CITED2, MMP-1, and MMP-13 (all from Abcam), followed by incubation with AlexFluor 488 conjugated anti-mouse (for acetylated a-tubulin staining) or antimouse or anti-rabbit secondary antibody (Biocare Medical) and visualization with 3,3ʹ-Diaminobenzidine (DAB) chromagen (Vector Laboratories). Negative controls were stained with irrelevant isotype-matched antibodies (Biocare Medical). Cilia were counted by scoring acetylated a-tubulin-positive structures greater than 1 mm in length from at least five different views of each section 27 .
Percentages of positively stained chondrocytes were determined by counting the number of immunostained cells and dividing by the total number of chondrocytes visualized by a hematoxylin counterstain (Vector Laboratories).