Dab substrate kit

Sections of formalin‐fixed, paraffin‐embedded tissues, xenografts, and ALI organotypic cultures were stained by standard HE staining. For immunohistochemistry staining, slides were subjected to antigen retrieval in citrate buffer (pH 6.0, Sigma‐Aldrich) at 95 °C for 20 min, and a blocking procedure was performed overnight with 5% bovine serum albumin (Sigma‐Aldrich,) and 0.05% Triton X‐100 (Sigma‐Aldrich) in DPBS(‐) (Gibco) at 4 °C. Primary antibodies used in this study included antibodies against MUC2 (Santa Cruz Biotechnology, SC‐15334, 1:500), KI67 (Thermo Fisher Scientific, 550609, 1:1000). Then, sections were stained using DAB Substrate Kit (#550880, BD). Images were acquired by Olympus IX73 microscopy.

Bone tissue sections were rehydrated through xylene and graded ethanol, incubated in the proteinase K (20 μg/ml) for 10 min and then sections were blocked for endogenous peroxidase with 3% hydrogen peroxide (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min in room temperature. Sections were permeabilized and blocked in 10% goat serum for 1 hr. The primary antibodies were anti-collagen I (1:600, ab138492 and ab21286, Abcam, Cambridge, MA, USA) diluted in the 5% goat serum and incubated at 4°C overnight. Sections were then incubated with a biotinylated goat anti-rabbit antibody (BD Pharmingen, San Diego, CA, USA). For detection, Streptavidin-Horseradish Peroxidase and DAB Substrate Kit (BD Pharmingen, San Diego, CA, USA) were used and the counterstain was done with hematoxylin. For osteocalcin (AbD Serotec 7060–1815, 1:1200 dilution), androgen receptor (Santa Cruz, sc-816, 1: 200) and ERβ (Santa Cruz, sc-8974) antigen retrieval was done with 0.5% trypsin at 37°C for 30 min and for estrogen receptor alpha (Santa Cruz 8002, 1:150), antigen retrieval was done in 10 mM sodium citrate, pH 6, at 95°C for 15 min.

6-Hydroxydopamine hydrochloride and ascorbic acid were purchased from Sigma-Aldrich (St. Louis, MO) and pioglitazone (Actos TM) was purchased from Takeda Pharmaceuticals (Osaka, Japan). Antibodies targeting PGC1α, TFAM, Nrf2 and catalase as well as ECL substrate were obtained from Santa Cruz Biotechnology (Dallas, TX), Mitobiogenesis Western Blot cocktail, anti-TH and anti-β-actin (1,1000) from Abcam (Cambridge, MA), Entellan from Merck (Darmstadt, Germany) and a DAB substrate kit from BD Biosciences (San Jose, CA). Pioglitazone tablets were crushed into powder and dissolved in 0.9% physiological saline for intragastric administration.