Citric acid ph 6

Paraffin-embedded skin specimens were prepared using routine methods. The sections were stained with hematoxylin and eosin. Immunohistochemistry was performed as previously described with some modifications^{10 (link)}. In brief, sections were deparaffinized and hydrated by washing in xylene and treatment with graded alcohol series. To unmask antigens, sections were incubated in 10 mM citric acid (pH 6) (Dako) at 95 °C for 30 min. Sections were then blocked with normal serum for 60 min at room temperature and then treated with primary antibodies (S100A2; Abcam, Cambridge, UK). Samples were washed and incubated for 30 min with secondary antibodies and visualized by staining with 3,3-diaminobenzidine.

Immunohistochemical staining for STING was conducted as previously described, though with several modifications^{34 (link)}. In brief, section specimens were deparaffinized and hydrated by xylene washing and treated with a graded series of alcohol. For treatment with unmasking antigens, section specimens were treated with 10 mM citric acid (pH 6) (Dako) at 95 °C for 30 min and then blocked with normal sera at room temperature for 60 min. The specimens were then incubated with a primary antibody (STING; Proteintech, Wuhan, China). After washing the samples, the sections were incubated at room temperature for 30 min with secondary antibodies, and the targeted antigens were visualized by 3,3-diaminobenzidine staining. To evaluate the degree of STING-positive cells in the sections precisely, the frequencies of immunoreactive lymphocyte were counted in each immunostaining section. Tumors containing more than 40% of STING-expressing cells were classified as the high STING-expression group, while those containing less than 40% of STING-expressing cells were classified as the low STING-expression group.