Techmate 500

For immunohistochemical evaluation of all the tumours we constructed tissue microarrays (TMAs). We selected a minimum of 2 areas per tumour and a total of 4 TMAs blocks were performed. Each TMA block was cut into four micrometer sections.
Immunohistochemical studies were performed from tissue microarrays with the automated immunohistochemical system TechMate 500^® (Dako Co, Carpinteria, CA), using the EnVision system (Dako). Briefly, 4 µm sections were deparaffinized and hydrated through graded alcohols and water. Peroxidase was blocked for 7.5 minutes in ChemMate peroxidase-blocking solution (Dako). Then, the slides were incubated with the primary antibodies for 30 minutes and washed in ChemMate buffer solution (Dako). The peroxidaselabelled polymer was then applied for 30 minutes. After washing in ChemMate buffer solution, the slides were incubated with the AEC substrate chromogen solution, washed in water, counterstained with hematoxylin and mounted. The primary antibody used in the study was Survivin (Abcam, Cambridge, UK; 1/500 dilution).

Immunostaining was performed on an automated staining system (TechMate 500, Dako, Hamburg, Germany) using the streptavidin-biotin complex method. Antibodies included the mouse anti-human Ki-67 IgG₁ monoclonal antibody (clone MIB-1, dilution 1:1000, Dako) and the CINtec^® Histology kit for the evaluation of p16^INK4a (Roche, Basel, Switzerland).

Tumor grading, tumor typing and immunohistochemistry (ER, PR, Ki-67) was performed on the pretreatment core biopsies on all patients. Pathological complete response (pCR) was determined on tumor resection specimens after completion of neoadjuvant chemotherapy, and was defined as no evidence of residual invasive and ductal disease in the breast and lymph nodes (ypT0,ypN0).
Immunohistochemistry was performed according to previously standardized protocols on an automated IHC platform (Dako Techmate 500) with citrate buffer for antigen retrieval [23 (link)] and observing the ASCO/CAP guidelines for immunohistochemistry [7 (link)]. The following primary antibodies and corresponding dilutions were used (DakoCytomation, Glostrup, Denmark): ER (clone 1D5, 1:100), PR (clone PgR636, 1:100) and Ki-67 (MIB-1, 1:200). Slides were assessed by quantitative image analysis (qIHC) using the Aperio Image Analysis toolbox (Leica Biosystems, Nussloch, Germany). Staining intensity and percentage of positive nuclei were recorded after manually segmenting tumor from adjacent stroma. Tumors with ER/PR Remmele scores greater than 3 or positive nuclei greater than 1% were considered hormone receptor positive.

Three-micrometer slices were sectioned from the TMA block and applied to coated, immunochemistry slides (DAKO, Glostrup, Denmark). The slides were baked overnight in a 56°C oven, deparaffinized in xylene for 20 min, rehydrated through a graded ethanol series and washed with PBS. A heat-induced epitope retrieval step was performed by heating a slide in a solution of sodium citrate buffer pH 6.5 for 2 min in a conventional pressure cooker. After heating, the slides were incubated with proteinase K for 10 min and rinsed in cool running water for 5 min. Endogenous peroxide activity was quenched with 1.5% hydrogen peroxide (DAKO) in methanol for 10 minutes, and incubation with the primary antibodies anti-gamma H2A.X (phospho S139) antibody (ab11174 from Abcam) and anti-p53: p53 FL 393 (sc-6243 from Santa Cruz); was performed for 40 min. After incubation, immunodetection was performed with the EnVision (DAKO, Glostrup, Denmark) visualization system using diaminobenzidinechromogen as the substrate, according to the manufacturer's instructions. Immunostaining was performed in a TechMate 500 automatic immunostaining device (DAKO) and measured through a double-blind visual assessment using microscopic observation according to the anatomopathological experience of pathologists. Sample scoring was performed by microscopic analysis, considering the percentage of nuclei stained cells.