Antigen unmasking solution

Formalin fixed, paraffin embedded adipose tissue samples were prepared and used to make sections for immunohistochemical studies as described previously24 (link). Briefly, sections were deparaffinized and the antigens were retrieved at high-temperature using antigen unmasking solution (Dako, Denmark). The endogenous peroxidase was quenched using 3% H₂O₂ (Merck Schuchardt, Gemany) for 60 min at RT. Sections were blocked with 5% fat-free milk for 60 min at RT followed by 1% BSA for another 60 min and then, incubated at 4 °C for overnight with primary antibodies as mentioned in the Figures. After washing, sections were stained with horseradish conjugated secondary antibody (Dako, Denmark) for 60 minutes at RT. Colors were developed using DAB kit (Dako, Denmark) and sections were counterstained with hematoxylin (Sigma Aldrich, St. Louis, MO). Quantification of the immunohistochemical staining data was done using Aperio software version 6.3 (Molecular Devices, Downingtown, PA) with an established arbitrary threshold.

Mouse femur and tibia were fixed in 10% neutral-buffered formalin, decalcified in 12% EDTA and embedded in paraffin. Sections (5 μm) were stained with hemotoxylin & eosin to evaluate morphology. For 4-HNE immunohistochemistry, bone sections were de-paraffinized, heated in antigen unmasking solution (Dako, Carpinteria, CA) and permeabilized in phosphate-buffered saline (PBS, pH 7.4) containing 0.2% Triton-X-100. Sections were blocked in 1% bovine serum albumin (BSA) for 1 h, incubated with primary antibody (Alpha Diagnostics, San Antonio, TX) overnight at 4°C, followed by incubation in peroxidase-conjugated antibody (Vectastain Elite Reagent, Vector Labs, Burlingame, CA) and in Sigma Fast 3,3′-diaminobenzidine substrate (Sigma, St. Louis, MO). Sections were counterstained with 0.1% Methyl Green in PBS and examined by light microscopy. For quantification, three sections per mouse (n = 3/group) were immunostained twice and analyzed using Image J. Briefly, channels were separated, the blue channel image was inverted and the threshold adjusted to remove nuclear background. Average staining intensity and stained cell counts were measured in several ROI of the same size.

Assessment of the histopathology of IP and IB STOSE tumors was performed by staining sections with hematoxylin and eosin (H and E) and by immunohistochemical analysis. Following deparaffinization in xylenes and rehydration in an ethanol gradient, antigen unmasking (antigen unmasking solution, Dako) was performed, followed by blocking endogenous peroxidase activity using 3% hydrogen peroxide in dH₂O. Sections were then rinsed in PBS. Immunostaining for mouse cytokeratin (pan-CK; pre-diluted, Abcam), mouse WT1 (1:100, Dako), and mouse inhibin (1:100, Dako) was performed according to the mouse-on-mouse kit (Vector). Immunostaining for rabbit PAX8 (1:400, Santa Cruz Biotechnology) was done by incubating sections with the PAX8 antibody overnight at 4^oC, followed by anti-rabbit horseradish peroxidase-labeled polymer (Dako) for 30 min at room temperature. All sections were counterstained using hematoxylin and developed using diaminobenzidine. Following dehydration in an ethanol gradient, sections were mounted using Permount (Fisher Scientific). Images were acquired using the ScanScope CS2 (Aperio).