Dako autostainer plus system

IHC staining was performed using a Dako Autostainer Plus system (Dako; Agilent Technologies, Carpinteria, USA). Tissues were pre-treated strictly according to the requirements of the manufacturer of the Dako Autostainer Plus system, including de-paraffinization and rehydration, and were subjected to 5-min pressure-cooking antigen retrieval, 15-min endogenous enzyme blocking, 60-min primary antibody (TN-C antibody, 1:300. cat: Sc-25,328; Santa Cruz Biotechnology, Inc., Texas U.S.A.) incubation at room temperature and a 30-min incubation at room temperature with Dako Cytomation EnVision-HRP reagent rabbit antibodies (1:200; Dako; Agilent Technologies, Carpinteria, USA). Signals were measured according to substrate hydrogen peroxide using DAB as a chromogen followed by hematoxylin counterstaining. Negative controls were prepared by omitting the primary antibody. Stained (brown) cells were quantified by counting the positive cells × 100/total cells in 10 random microscopic (Olympus, Japan) (× 400) fields in each section.

A mouse monoclonal antibody against SDC1/CD138 (clone MI15, dilution 1:100, Dako/Agilent, Santa Clara, CA, USA) was used to perform IHC staining after heat-based antigen retrieval (30 min, 96°C, pH 6.0) on the 4 µm thick FFPE sections. Automated IHC was performed using the Dako Autostainer Plus System (Dako) with the anti-mouse IgG EnVision Plus detection kit (Dako) for secondary and tertiary immunoreactions. Negative controls were calculated by each run [20 ]. Reaction products were developed with diamino-benzidine (DAB), according to general protocols. SDC1 staining intensity was scored as 1, 2, or 3, equivalent to negative, moderate, and strong intensities. A percentage score was also defined as 0–10%—0 Pts., 11–20%—1 Pt., 21–30%—2 Pts., 31–40%—3 Pts., 41–50%—4 Pts., 51–60%—5 Pts., 61–70%—6 Pts., 71–80%—7 Pts., 81–90%—8 Pts., 91–100%—9 Pts. Finally, a score was calculated by multiplying the intensity score and percentage score. Moderate SDC1 expression was considered as a score between 4 and10, and strong expression was considered as a score higher than 12. SDC1 expression was evaluated separately for cell membrane, cytoplasm, and stroma.

Mouse prostate tissues and clinical specimens were stained with antibodies specific for DAB2IP, p-STAT3 (Y705) and survivin using Dako Autostainer Plus system (Dako, Carpinteria, CA, USA) as described.^{11 (link)} The expression of DAB2IP, p-STAT3 (Y705) and survivin was scored based on the percentage and intensity according to Allred's scoring protocol.^{39 (link)} Values on a four-point scale were assigned to each specimen. The intensity score was assigned, which represented the average intensity of positive cells (0, none; 1, weak or questionably present stain; 2, intermediate intensity in a minority of cells; and 3, strong intensity in a majority of cells). All slides were scored independently by two investigators who were blinded to patient clinical information.