[CITATION][C] Mutational analysis of E‐cadherin, β‐catenin and APC genes in synovial sarcomas

MM Subramaniam, S Calabuig‐Fariñas… - …, 2010 - Wiley Online Library
MM Subramaniam, S Calabuig‐Fariñas, A Pellin, A Llombart‐Bosch
Histopathology, 2010Wiley Online Library
Details of immunohistochemistry (IHC): antigen retrieval consisted of two techniques:*(i)
heat-induced epitope retrieval consisting of steaming in citrate buffer (pH 6.0) in a pressure
cooker at 1.5 ATM pressure for 3 min;|(ii) enzyme treatment in the form of incubation of tissue
sections in Tris-ethylenediamine tetraacetic acid (EDTA) buffer pH 8.0 for 20 min. For all the
antibodies, the incubation time was 1 h. Antigen–antibody complexes were visualized using
a biotin–streptavidin detection system (ChemMate Detection kit; Dako, Glostrup, Denmark) …
Details of immunohistochemistry (IHC): antigen retrieval consisted of two techniques:*(i) heat-induced epitope retrieval consisting of steaming in citrate buffer (pH 6.0) in a pressure cooker at 1.5 ATM pressure for 3 min;|(ii) enzyme treatment in the form of incubation of tissue sections in Tris-ethylenediamine tetraacetic acid (EDTA) buffer pH 8.0 for 20 min. For all the antibodies, the incubation time was 1 h. Antigen–antibody complexes were visualized using a biotin–streptavidin detection system (ChemMate Detection kit; Dako, Glostrup, Denmark) with 3, 3-diaminobenzidine (DAB; Fluka Chemie GmbH, Steinheim, Germany) as chromogen. Non-immune mouse and rabbit serum samples were substituted for the primary antibodies as negative controls. Appropriately validated positive controls were run simultaneously for all the tested antibodies. In biphasic synovial sarcomas (BSSs), the glandular component and stromal spindle cell component were scored separately and when there was preserved expression in one of the components it was scored accordingly. IHC scoring: E-cadherin⁄ a, b and c catenins were scored as described previously. 1Dysadherin immunoreactivity was classified as follows: 0–9%(1+, sporadic), 10–29%(2+, focal), 30–49 (3+) and‡ 50%(4+, diffuse). Tumours were considered dysadherin-positive when more than 50% tumour cells were positive. Snail⁄ Slug immunoreactivity was scored as follows: 0, no reactivity or< 1% positive tumour cells, 1+, 1–4% positive tumour cells; 2+,> 5–10% positive tumour cells and 3+,> 10% positive tumour cells. Positive reaction was defined as only nuclear reactivity. AKT, phosphorylated AKT (pAKT), phosphorylated glycogen synthase kinase 3beta (GSK3 b), phosphorylated GSK3 b (pGSK3 b) and phosphotidylinositol 3¢ kinase (P13K) was scored on a three-point scale based on staining intensity: mild, moderate and intense.
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