Quantitative Immunohistochemistry of 14-3-3η in Synovial Tissue

Formalin-fixed, paraffin-embedded synovial tissue sections (5 µm) were deparaffinized and rehydrated. Immunohistochemical staining was performed according to the standard avidin–biotin immunoperoxidase complex technique [4 (link)], and the sections were incubated with anti-14-3-3η (Augurex, 1:100 in 2% BSA and 2% goat serum) or mouse isotype IgG (DAKO Agilent, Santa Clara, CA, USA,1:100). Diaminobenzidine was used as a substrate for the detection of the labeled proteins, and the sections were counterstained with Harris hematoxylin. Slides were scanned using a Hamamatsu Nanozoomer 2.0-RS scanner. For quantitative immunohistochemistry, six random fields (at original magnification 20X) for each patient were captured using NPD viewing software, and the intensity of labeling in the tissue sections was analyzed using the immunohistochemistry quantification technique as previously described [5 (link)]. The results are expressed as the sum of labeling intensity (density) relative to the total area.