Immunohistochemistry steps were performed by standard protocols. Antigen retrieval was conducted by immersing slides in Tris-EDTA buffer (pH 9.0) at 120°C for 3 min, and primary mAb RP215 (3.99 mg/ml) was provided by Xiaoyan Qiu of Peking University (the cell line of monoclonal antibody RP215 was generated by Gorgey Lee of the University of British Columbia in Vancouver, Canada), and the dilution was 1:3000. Secondary antibodies were from Jackson Immuno Research and Invitrogen, antigen was visualized with substrate chromogen (Dako liquid DAB chromogen; Dako), staining was performed according to the instructions. The expression of SIA-IgG was qualified by a four-tier intensity score (0, none; 1, weak; 2, moderate; 3, strong) and the percentage (0–100%) of positive cells. The final staining score was obtained by multiplying the intensity and percentage of positive cells (range 0–300) according to the percentage of positive cells present among all tumor cells. A score ≥150 was defined as high SIA-IgG expression, while scores <150 were defined as low SIA-IgG expression.
Dako liquid dab chromogen
Manufactured by Agilent Technologies
Dako liquid DAB chromogen is a laboratory product used for the detection and visualization of specific target molecules in tissue samples during immunohistochemical procedures. It provides a brown chromogenic reaction when applied to the sample.
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2 protocols using dako liquid dab chromogen
1
Immunohistochemical Evaluation of SIA-IgG Expression
2
Immunohistochemical Analysis of LSD1 Expression in Human Liver Tissues
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The expression patterns of LSD1 in human liver tissues were examined by immunohistochemistry as described previously [30 (link),31 (link),32 (link),33 (link)]. Briefly, slides of paraffin-embedded liver tumor specimens were processed under high pressure (125 °C, 30 s) in antigen-retrieval solution, high pH 9 (S2367, Dako, Carpinteria, CA, USA), treated with peroxidase blocking regent, and then treated with protein blocking regent (K130, X0909, Dako). Tissue sections were incubated with the rabbit anti-LSD1 polyclonal antibody (ab17721, abcam, Cambridge, UK; dilution used in IHC: 1:200), followed by HRP-conjugated secondary antibody (Dako). Antigen was visualized with substrate chromogen (Dako liquid DAB chromogen; Dako). Finally, tissue specimens were stained with Mayer hematoxylin (Hematoxylin QS; Vector Laboratories, Burlingame, CA, USA) for 20 s to discriminate the nucleus from the cytoplasm.
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