Hrp en vision

Immunohistochemistry was performed on 4–5 μm standard tumor tissue sections of formalin‐fixed paraffin‐embedded TMAs. All slides were dewaxed with xylene/ethanol before antigen retrieval in a pressure cooker (Decloaking Chamber Plus, Biocare Medical, Concord, CA, USA), or in a microwave, in Target Retrieval Solution (TRS) buffer (pH 9) (S2367; DAKO/Agilent). An endogenous block was applied prior to incubation with the primary antibodies to block for endogenous enzymatic activity. The slides were then incubated for 1 h with mouse monoclonal CD3, CD4, CD8, CD45, and FOXP3 antibodies (A0452 [Dako/Agilent, Santa Clara, CA, USA], 104‐R15 [Cell Marque, Darmstadt, Germany], M7103 [Dako], M0701 [Dako], and M560044 [BD Biosciences, San Jose, CA, USA], respectively) followed by incubation with an appropriate HRP‐En Vision (Dako), EnVision Mouse hrp, K4001 for CD8 and FOXP3, EnVision rabbit HRP, and K4003 for CD3 and CD4. Primary antibodies were omitted for the negative controls. Tissue from differentiated breast cancer types were used as positive controls.

Tissue sections of 4–5 μm were stained with primary antibodies for HSP27, VEGF-A, VEGF165b and bFGF. Furthermore, double staining with anti-factor VIII (F-VIII) and Ki67 was performed for angiogenesis assessment. After deparaffinizing in Xylene and different alcohol dilutions and rehydration, heat mediated or enzymatic antigen retrieval was performed. Endogenous peroxidase and alkaline phosphatase were blocked before incubation with the primary antibody followed by incubation with appropriate HRP-EnVision (DAKO, K4011 or K4007). For staining with HSP27 a secondary rabbit anti-goat antibody (Southern Biotech, Cat. no. 6164–01) was used; for double staining, a secondary goat anti-mouse antibody (Southern Biotech, Cat. no. 1031–04) was used. Details are provided in Table 1. For negative controls, primary antibodies were omitted or specific blocking peptides for HSP27, VEGF-A and bFGF were added. Tissues from different cancer types were used as positive controls.