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Before staining, coded ST samples were cut with a cryostat (5 μm), fixed with acetone and endogenous protease activity was blocked with 0.3% hydrogen peroxide.
After immunohistochemical staining, coded sections stained for CD135, CD68 or CD163 were analyzed in a random order by computer-assisted image analysis [ 28].
The association between Trop-2 immunohistochemical staining, coded as 0, 1, 2 and 3, and clinical covariates were evaluated by non-parametric one-way ANOVA and Wilcoxon-Mann–Whitney tests.
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We used the histological evaluation, i.e., Mankin scoring by three investigators from Safranin O stained blind coded sections, to evaluate microstructural integrity of articular cartilage.
Biomarkers were dichotomised (1=positive, 0=negative) to create binary variables with positive and heterogeneous staining being coded as positive and occasional or negative being coded as negative.
After immunohistochemical staining, all coded sections were randomly analyzed by computer-assisted image analysis (Fig. 1).
Fixed MSNs were numbered according to different conditions and coded before staining to ensure that the operator was blinded during the subsequent processing and analysis of immunofluorescence.
Stained sections were coded and randomly analyzed.
Stained slides were coded so that a blinded analysis could be performed.
Stained sections were coded and analyzed for the presence of necrobiosis by 2 independent observers (CD and JZ).
Stained sections were coded and analyzed by two independent observers, who were blinded to the diagnosis and clinical data.
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