Spectral dapi solution

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).