Immunohistochemical Analysis of FFPE Tissues

Formalin-fixed paraffin-embedded tissues (FFPE) and tumor microarray (TMA) sections (5 µm thickness) were deparaffinized in xylene and rehydrated in descending alcohols, followed by blocking of endogenous peroxidase in 3% hydrogen peroxide solution (30 min) and heat-induced epitope retrieval in citrate buffer (95 °C, 5 min). Sections were incubated with CD206 (1:100, Clone: C-10, Santa Cruz), iNOS (1:100, Clone: C-11, Santa Cruz), CD16b (1:100, Clone: CLB-gran11.5, BD Biosciences), Ly6G (1:100, Clone: 1A8, BioLegend), p-Smad3 (1:100, Clone: 1D9, Santa Cruz), p-Smad3 (1:200, 600-401-919, Rockland) antibodies overnight (4 °C), sections were incubated in polymer-HRP conjugated secondary antibody (Dako) for 2 h at room temperature, followed by DAB (Thermo-fisher) or Opal-520, 570, 650, 690 TSA dye (Akoya biosciences) development. DAB-stained section images were captured by the Nikon Ni-U Light Microscope and analyzed using Image J analysis software; Opal TSA-stained sections were captured on the Mantra quantitative pathology workstation (Akoya Biosciences) and analyzed by inForm image analysis software 2.6 (Akoya Biosciences) as per our previous studies^{18 (link),51 (link)}.