In Vivo Evaluation of the Antitumor and Immunogenic Properties of Silver and Sodium Dichloroacetate Combination against Melanoma

Figure 3

Immunocytochemistry of B16F10 cells. B16F10 cells were cultured in 6-well plates ( cells/well) on glass cover slips at 37°C and 5% CO₂ for 24 hours. Then, cells were treated with Ag (mM), DCA (195mM), or (mM and 135mM, respectively) for 4 hours and fixed with 100% methanol. Primary antibodies for HMGB1, HSP70, and HSP90 were applied, followed by HRP secondary antibody and ABC substrate kit (Vector Laboratories, Burlingame, CA). The cells were counterstained with hematoxylin (Vector), mounted onto slides, and imaged at 40x. Positive protein expression is evidenced by the presence of brown stain in the cell nucleus, cytoplasm, or both and quantified with the image analyzer software Fiji (ImageJ) version 2.0. Representative images of three independent experiments are shown (a). There is no statistical difference () between bars labelled with the () in the graph (b).

(a)

(b)