Pretreatment module

Tumour tissue microarray blocks were freshly cut into 4-μm sections. After deparaffinization in xylene and rehydration through a gradually decreasing concentration of ethanol to distilled water, slides were treated in a PreTreatment module (Lab Vision Corp., Fremont, CA, USA) in Tris–HCl (pH 8.5) buffer for 20 min at 98°C for antigen retrieval. Staining of sections was performed in an Autostainer 480 (Lab Vision) by the Dako REAL EnVision Detection system, Peroxidase/DAB+, Rabbit/Mouse (Dako, Glostrup, Denmark). Tissues were incubated with the mouse mAb HES9, at dilution of 1:500 (=5 μg/ml) for one hour at room temperature. A sample of renal tissue served as a positive control in each staining series.

The tumor tissue microarray (TMA) blocks were freshly cut into 4-µm sections, fixed on slides, and dried at 37 °C for 12 to 24 h. Then continued by deparaffinization in xylene and rehydration through a gradually decreasing concentration of ethanol to distilled water, TMA-slides were treated in a PreTreatment module (Lab Vision Corp., Fremont, CA, USA) in Tris-HCL buffer for 20 min at 98 °C for antigen retrieval. We stained sections with Autostainer 480 (LabVision) using Dako REAL EnVision Detection System, Peroxidase/DAB+, Rabbit/Mouse (Dako, Glostrup, Denmark).
First we treated slides with 0.3% Dako REAL Peroxidase-Blocking Solution for 5 min to block endogenous peroxidases. Then we incubated slides with our APCN antibody (1:1000 diluted in Dako REAL Antibody Diluent) for 60 min, followed by a 30 min incubation with peroxidase-conjugated Dako REAL EnVision, Rabbit/Mouse (ENV) reagent. Visualization of slides was by Dako REAL DAB + Chromogen for 10 min. Between every step in the staining procedure, slides were washed with 0.04%-Tween20 in PBS. We counterstained slides with Meyer’s hematoxylin, washed in tap water for 10 min, and mounted in an aqueous mounting medium (Aquamount, BDH, Poole, UK). Breast tissue with invasive lobular cancer served as a positive control.