All tissue samples were fixed in 10% formalin and sent to pathology for analysis. Routine hematoxylin and eosin staining was carried out in all samples either to confirm the diagnosis of polyps or to date the endometrium. Samples were embedded in paraffin blocks, cut into 4-μm-thick sections, and deparaffinized. The sections were then stained with primary monoclonal antibodies against COX-2 (1:100, clone CX-294, Dako, Glostrup, Denmark) and galectin-3 (1:100, NCL-GAL3, clone 9C4, NovaCastra, Hamburg, Germany) using a Dako Cytomation Autostainer (Dako). After staining, each sample was evaluated under a light microscope (200×, Olympus BX53, Olympus, Tokyo, Japan) to determine the percentage of COX-2–positive cells, the intensity of COX-2 staining, and the percentage of galectin-3–positive cells. For positive controls, staining of breast carcinoma tissue for COX-2 and papillary thyroid carcinoma tissue for galectin-3 were used. Primary monoclonal antibodies were omitted in negative controls.
Dako cytomation autostainer
Manufactured by Agilent Technologies
Sourced in Denmark
The DAKO Cytomation autostainer is a laboratory automation system designed for immunohistochemistry and in situ hybridization staining procedures. It automates the pretreatment, staining, and counterstaining steps of the staining process, providing consistent and reproducible results.
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5 protocols using dako cytomation autostainer
1
Immunohistochemical Analysis of COX-2 and Galectin-3 in Tissue Samples
2
Immunohistochemical Evaluation of MGMT Expression
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Formalin-fixed, paraffin-embedded sections were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. After blocking of endogenous peroxidase with 3% H2O2, the sections were pretreated in a microwave oven with a Tris-EGTA buffer and immunostained on a DAKO Cytomation autostainer (DAKO, Glostrup, Denmark), using monoclonal mouse anti-human antibody against MGMT (MAB16200, 1:200, Millipore). Immunoreactivity was visualized with DAB + (DAKO K3468) as chromogen. The immunohistochemical reactions were semiquantitatively evaluated according to the number of tumor cells stained: 0 = 0; 1%–10% = 1+; 11%–25% = 2+; 26%–50% = 3+; and >50% = 4+. For MGMT evaluation, positive endothelial cells, lymphocytes, and microglia served as positive internal controls.
3
Tissue Microarray Analysis of EZH2 in Metastatic Breast Cancer
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A tissue microarray (TMA) with two 1 mm cores from each of 220 MBC tumors was constructed as described (Nilsson et al., 2011 ). Sections of 3–4 μm were cut, transferred to SuperFrost Plus slides, dried at room temperature and then baked for 2 h at 60 °C. The DAKO Envision horseradish peroxidase rabbit/mouse kit (DAKO, Glostrup, Denmark) and a Dakocytomation Autostainer (DAKO) were used for the staining procedure. The TMAs were stained with a purified mouse anti‐EZH2 monoclonal antibody (clone 11, BD Transduction Laboratories, Franklin Lakes, NJ) at a 1:25 dilution after antigen retrieval at high pH as described elsewhere (Holm et al., 2012 ). EZH2 staining was scored by one reader (IJ) in a blinded manner. The percentage of positively stained tumor cells was evaluated and scored as: 0 (0%), 1 (1–10%), 2 (11–25%), 3 (26–50%), 4 (51–75%) or 5 (>75%). Tumors were considered positive for EZH2 if >50% of the cancer cells showed nuclear staining (Supplementary Figure S2 ).
4
MGMT Immunohistochemistry Assay Protocol
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MGMT immunohistochemistry assay was retrospectively performed on formalin-fixed, paraffin-embedded sections, which were deparaffinised in xylene and rehydrated in increasing concentrations of ethanol. After blocking of endogenous peroxidase with 3% H2O2, the sections were pre-treated in an oven with EnVision Flex TRS buffer and immunostained on a DAKO Cytomation autostainer (DAKO, Glostrup, Denmark), using monoclonal mouse anti-human antibody against MGMT (clone MT3.1, MAB16200 EMD MilliporeTM, Billerica, MA, USA). Immunoreactivity was visualized with DAB+ (DAKO K3468) as chromogen. The immunohistochemical reactions were semiquantitatively evaluated according to the number of tumor cells stained. Subgroup analyses were exploratory performed using three cut-off of MGMT expression: negative vs positive, < vs ≥ of 50% of cells stained, and < vs ≥ 70% of cells stained.
5
Tumor-Specific CYP27A1 Expression Analysis
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The tumor cell-specific expression of CYP27A1 was determined by immunohistochemistry following a previously validated protocol (Nelson et al. 2013) . Sections of 3 to 4 µm were cut from whole tissue FFPE blocks, de-paraffinized, treated with antigen retrieval buffer (citrate, pH 6) for 20 min, and then reacted with an anti-CYP27A1 rabbit monoclonal antibody (ab126785, Abcam) at a dilution of 1:500 for 2 h. Staining procedures were performed using the DAKO Envision horseradish peroxidase rabbit/mouse kit (DAKO) and the Dakocytomation Autostainer (DAKO). Cell nuclei were counterstained with hematoxylin. CYP27A1 positivity was detected as a granular cytoplasmic reactivity. Cell type identification and staining intensity was assessed by a board certified pathologist (DG). Only tumor cell-specific CYP27A1 expression was considered for subsequent analyses. Each sample was given a semiquantitative intensity score: 0 (absent), 0.5 (borderline), 1 (weak), 2 (moderate) or 3 (strong). For statistical analysis, the tumors were categorized as negative (0), weak (0.5, 1) and overexpressed (2, 3). Representative cases of CYP27A1 expression in these categories are illustrated in Supplementary Fig. 4.
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