Anti cd1c

An independent set of formalin-fixed paraffin-embedded tissue specimens, namely, 3 cases of NC, and 3 cases of THYM, were obtained from the Department of Pathology of the first affiliated hospital, at Jinan University and used for immunohistochemical analysis. Immunohistochemistry was performed according to the procedure. Briefly, 4-μm-thick tissue sections were deparaffinized, rehydrated, and treated with an antigen retrieval solution (10 mmol/L sodium citrate buffer, pH 6.0). The sections were incubated with anti-CD1C (1:250; Abcam) overnight at 4°C and then incubated with biotinylated secondary antibody followed by addition of avidin-biotin peroxidase. Finally, tissue sections were incubated with 3′,3′-diaminobenzidine until a brown color developed, and they were counterstained with Harris’ modified Hematoxylin. In negative controls primary antibodies were omitted.

The experimental process of specimen preparation was the same as for a single IHC. Primary antigen retrieval was performed using High pH Target Retrieval Solution (DAKO, Glostrup, Denmark). The primary antibodies were anti-CD1c (mouse monoclonal, clone OTI2F4; Abcam, Cambridge, UK), anti-CD4 (mouse monoclonal, clone 4B12; Invitrogen, Waltham, USA), anti-CD8 (mouse monoclonal, clone C8/144b; DAKO, Glostrup, Denmark), anti-CD19 (mouse monoclonal, clone LE-CD19; Invitrogen, Waltham, USA), anti-CD21 (rabbit monoclonal, clone SP32; Abcam, Cambridge, UK), and anti-CD138 (mouse monoclonal, clone MI15; DAKO, Glostrup, Denmark). For secondary detection, we used a horseradish peroxidase-labelled detection system (EnVision plus; DAKO, Glostrup, Denmark) as a catalyst for fluorophore-conjugated tyramide. Antigen stripping was performed using Immunoactive Retrieval Buffer (pH 6; Matsunami Glass, Osaka, Japan). Tyramide signal amplification was performed using Opal fluorophore reagents (Akoya Biosciences, Marlboro, USA); Opal 520, Opal 540, Opal 570, Opal 620, Opal 650, and Opal 690 were used for CD1c, CD4, CD8, CD19, CD21, and CD138, respectively. DAPI counterstaining was performed using Spectral DAPI solution (Akoya Biosciences, Marlboro, USA).