Quantifying AGE, SMA, and DPP4 in Aged Omental Tumors

Tumor bearing omenta from young and aged mice (n=13) [3 (link)] were fixed with 10% formalin, then paraffin embedded and sectioned (5μm). Sections were de-paraffinized by xylene and were rehydrated in decreasing concentrations of ethanol. Antigen retrieval was done with 10mM sodium citrate, pH 6.0 (SMA, AGE antibodies) or Tris-EDTA buffer (10mM Tris, 1mM EDTA, 0.05% Tween 20, pH 9.0; DPP4 antibody). Endogenous peroxidases were blocked with 3% H₂O₂. Slides were blocked in horse serum, then incubated overnight at 4°C with an anti-AGE antibody (Abcam, ab23722, 1:10,000), an anti-SMA antibody (Abcam, ab5694, 1:1000), or anti-DPP4 antibody (Abcam, ab187048, 1:20,000) diluted in horse serum. All antibodies were then incubated with ImmPRESS HRP Horse Anti-Rabbit IgG (Vector Labs MP-7401) according to manufacturer’s specifications, developed with 3,3-diaminobenzene and counterstained with hematoxylin. Tissues were dehydrated in increasing concentrations of ethanol followed by xylene and mounted with Cytoseal XYL. Slides were imaged with Aperio ScanScope CS and analyzed with ImageScope software.