M3635

Formalin-fixed and paraffin-embedded tumor tissue from core biopsies and surgical samples were cut into 3 to 4 μm sections and transferred to glass slides (Menzel Super Frost Plus), dried at room temperature, and baked in a heated chamber for 2 hours at 60^○C. De-paraffinization and antigen retrieval was performed using PT Link (Dako Denmark A/S) using a high pH buffer. Staining was performed in an Autostainer Plus (Dako) using a di-amino-benzidine (DAB) based visualization kit (K801021-2, Dako). Counterstaining was performed using Mayer’s hematoxylin with antibodies against ER (SP1, Thermo Scientific, diluted 1:200), PR (Dako M3569, diluted 1:200), HER2 (4B5, Ventan BenchMark Ultra, Ventana Medical Systems, Inc. Tucson, Arizona, R.U.), cyclin D1 (Dako M3635, diluted 1:40), and p27 (Dako M7203, diluted 1:100).

Stromal cyclin D1 was detected in clinical breast cancer specimens by immunofluorescence-immunohistochemistry (IF-IHC) performed on an Autostainer Plus (Dako) as previously described [87 (link)] with the following specifications: antigen retrieval used the DAKO PT-module with citric acid buffer (pH 6.0) and rabbit monoclonal cyclin D1 antibody (Dako, M3635) was diluted 1:200 and coincubated with mouse monoclonal anti-pancytokeratin (clone AE1/AE3, DAKO, 1:100) for 45 minutes.
Quantitative analysis of cyclin D1 was performed as previously described [86 (link)]{Peck, 2016 #603;Peck, 2016 #230} using the ScanScope FL line scanner (Leica Biosystems) to capture high-resolution digital images followed by quantification of cyclin D1 levels in the stroma of tumor specimens using Tissue Studio image analysis software (Definiens). Briefly, user-guided machine learning was performed to generate an analysis solution that identified DAPI-stained cell nuclei within the stroma of each tumor specimen. Mean nuclear cyclin D1 signal intensity was calculated for stromal cells in each tumor core.

Fine sectioning (5 μm) was done in formalin-fixed paraffin-embedded specimens on poly-L-lysine (Sigma, St. Louis, MO, USA) coated slides, followed by immunohistochemistry (IHC). The antigen retrieval for Cyclin D1 was performed in 10 mM tris-EDTA buffer (pH-9.0) at 900W for 15 min/360W for 5 min. The sections were consecutively washed in Tris buffer saline (TBS; 0.1M; pH: 7.4) and then incubated with primary antibody of Primary rabbit monoclonal of Cyclin D1 (monoclonal, M3635, Dako cytomation Glostrup, Denmark; 1:200 dilution) overnight followed by the use of polymer based secondary antibody (Envision System peroxidase kit, DAKO, Carpinteria, CA) for 1 hr at room temperature after washing with TBS. Previous studies from our laboratory have mentioned the complete methodological details^{9 (link),10 (link)}.