Diaminobenzidine tetrahydrochloride

Tissue samples were rinsed with PBS, fixed in 4% paraformaldehyde, dehydrated in a graded series of ethanol solutions, embedded in paraffin and sectioned (5 µm) on a microtome (RM2235, Leica). Sections were stained with hematoxylin and eosin (H&E) and Masson's trichrome stain. For immunohistochemical staining, sample sections were incubated with primary antibodies against CD31 (1∶200; Rabbit polyclonal antibody against mouse, CD31, Abcam), followed by horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibody (1∶200 in PBS containing 1% BSA, Santa Cruz) and color development with diaminobenzidine tetrahydrochloride (Santa Cruz). Nuclei were counterstained with hematoxylin. Images were taken on a phase contrast microscopy (Eclipse TS100, Nikon). To quantify the positive staining of CD31, for each sample, 3 sections were stained and for each section, 3 images were taken for analysis. Image J software was applied to determine the mean vessel density by calculating positively stained vessel area per unit area. Artifacts of “false” positive staining for microcarriers were manually excluded during the calculation.

Samples were fixed in 4% paraformaldehyde and embedded in paraffin for sectioning at 5 μm thicknesses and then mounted on glass slides. Sections were stained with hematoxylin and eosin (HE) and Safranin-O (SO).
For immunohistochemical analysis, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) to detect apoptotic cells was performed using a TUNEL kit (Roche, Indianapolis, IN, United States) in accordance with the manufacturer’s instructions. A rabbit anti-human monoclonal antibody against collagen II (COL II) was used with a horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (1:400 in PBS, Santa Cruz) as the secondary antibody. CD3 was detected using a rabbit anti-human CD3 monoclonal antibody (1:100 in PBS, Santa Cruz Biotechnology, Santa Cruz, CA, United States). CD68 was detected using a rabbit anti-human CD68 monoclonal antibody (1:1,000 in PBS, Santa Cruz Biotechnology). Color development was conducted with diaminobenzidine tetrahydrochloride (Santa Cruz Biotechnology).

Samples were cut into 6 μm sections after being fixed in paraformaldehyde and embedded in paraffin. Hematoxylin and eosin (H&E), safranin-O, Masson's trichrome, and Von Kossa staining were performed according to standard procedures. Two-step indirect immunohistochemical staining was performed to investigate the expression of type I, type II, and type X collagen in matrices using a primary anti-collagen type I antibody (mouse anti-rabbit, 1 : 50; Abcam, Cambridge, MA, USA), anti-collagen type II antibody (mouse anti-rabbit, 1 : 50; Acris, Herford, Germany), and anti-collagen type X antibody (mouse anti-rabbit, 1 : 50; Abcam, Cambridge, MA, USA), followed by a horseradish peroxidase-conjugated anti-mouse antibody (1 : 200 in PBS; Santa Cruz, Dallas, Texas, USA) and color development with diaminobenzidine tetrahydrochloride (Santa Cruz, Texas, USA). The sections were counterstained with hematoxylin solution (Mayer's).