IHC assays were performed as described [23 (link)], using a DAKO immunostainer (DAKO, Carpinteria, CA) with antibodies against c-Myc (N262, Santa Cruz Biotechnology, Santa Cruz, CA). 41 patients sample with gene expression data were used for the analysis. Archival formalin-fixed and paraffin-embedded tissues of breast cancer patients were obtained from the surgical pathology archive of the University of Chicago for tissue microarrays (TMA) construction. Cell lines HCC38 and UACC3199 were pelleted and processed by formalin-fixation and paraffin-embedding, then stained with c-Myc antibody and served as positive control. Immunostaining was scored semi-quantitatively by two observers (G. Khramtsova and A. Khramtsov). Scoring was based on intensity and percentage of positive stained cells (nuclei, cytoplasm and membrane respectively). All discrepancies were resolved by a second examination by two observers simultaneously using a multiheaded microscope. The results were scored using a scale from 0 to 3. Weak staining in <15% of tumor cell nuclei was considered as 0 (“negative”), whereas a score of 1 (“weak”) was assigned when >15% of the nuclei were weak positive. Scores 2 and 3 (strong immunoreactivity in ≥15% of the nuclei) were considered together as “strong” signal.
Dako immunostainer
Manufactured by Agilent Technologies
Sourced in United States
The DAKO immunostainer is a laboratory instrument designed for automated immunohistochemical (IHC) staining. Its core function is to perform standardized and reproducible IHC staining procedures on tissue samples.
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Lab products found in correlation
3 protocols using dako immunostainer
1
Immunohistochemical Analysis of c-Myc in Breast Cancer
2
Quantifying Cardiac α-SMA Positive Cells
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Immunohistochemical analysis for alpha-smooth muscle actin (α-SMA)-positive cells was performed 14 days after AMI development on formalin-fixed, paraffin-embedded cardiac tissue sections using a DAKO immunostainer (DAKO, USA) according to the manufacturer’s protocol. Sections (4 μm) were deparaffinized and rehydrated through graded alcohols. Endogenous peroxidase activity was blocked with 0.3 % hydrogen peroxide for 10 minutes, and antigens were retrieved for 20 minutes in 10 mmol/l citrate buffer, pH 6.0, at 98 °C. Nonspecific binding was blocked with 10 % normal goat serum for 20 minutes. The tissue slides were incubated with the antibody (1:500 dilution; DAKO) for 65 minutes at 25 °C. Then the slides were incubated with a ChemMate DAKO Envision system (K5007) for 30 minutes at 25 °C and subsequently reacted with 3,3′-diaminobenzidine as a chromogen substrate. The nucleus was counterstained using Mayer hematoxylin. A score from 0 to 3+ was applied; where 0 = absence of α-SMA positive cells in 5 fields, 1 = 1–5 cells/5 fields, 2 = 6–10 cells/5 fields and 3 = more than 10 cells/5 fields (magnification ×100).
3
Standardized Immunohistochemical Profiling
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Five-micron tissue sections were cut from the selected block. Immunostaining assays were performed on a Leica Bond Platform (Leica Microsystems, Chicago, IL, USA) for French cases and an automated DAKO Immunostainer for Italian cases.
The antibodies were used according to the manufacturer's protocol. The same panel of antibodies was applied to all cases. The antibodies/antisera used are listed in Table 1. A stain was considered 'positive' when at least 5% of neoplastic cells showed immunoreactivity in the appropriate site.
The antibodies were used according to the manufacturer's protocol. The same panel of antibodies was applied to all cases. The antibodies/antisera used are listed in Table 1. A stain was considered 'positive' when at least 5% of neoplastic cells showed immunoreactivity in the appropriate site.
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