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.
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.
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