Paraffin-embedded samples were cut into 5 µM sections. After being deparaffinized and hydrated, the sections in Tris-EDTA buffer (pH 9.0) were subjected to heat-induced antigen retrieval in a microwave oven, and then treated with 3% hydrogen peroxide. Following incubation with goat antihuman PD-1 antibody (R&D systems, Minneapolis, MN) overnight at 4°C, slides were treated with horseradish peroxidase-conjugated secondary antigoat antibody (Zhongshan Golden Bridge Biotech, Beijing, China) and then 3,3′-diaminobenzidine tetrahydrochloride. For colocalization analysis, after PD-1 staining, sections were stained with a mouse antihuman CD8 antibody (DAKO, Glostrup, Denmark), and visualized using EnVision G2 System/AP Rabbit/Mouse (PermanentRed) (DAKO). Slides were examined using a microscope (Nikon, Tokyo, Japan).
Envision g2 system ap rabbit mouse permanent red
Manufactured by Agilent Technologies
Sourced in Denmark
The EnVision G2 System/AP Rabbit Mouse Permanent Red is a laboratory equipment product from Agilent Technologies. The core function of this system is to perform immunohistochemistry (IHC) and immunocytochemistry (ICC) assays using a permanent red chromogen.
Automatically generated - may contain errors
4 protocols using envision g2 system ap rabbit mouse permanent red
1
Immunohistochemical Analysis of PD-1 Expression
2
Immunohistochemical Detection of PTEN
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The immunohistochemistry procedures were carried out according to the previous method with appropriate modifications [27 (link)]. The tissue sections were incubated with PTEN antibody (ab170941, 1:100) and stained with horseradish peroxidase (HRP) anti-rabbit immunoglobulin G and diaminobenzidine. Next, the sections were incubated with secondary antibody and stained with EnVision G2 System/AP Rabbit Mouse (Permanent Red) (Dako, Glostrup, Denmark). Lastly, the sections were counterstained with hematoxylin and observed under a fluorescence inverted microscope (Hitachi Limited, Tokyo, Japan).
3
Immunohistochemical Analysis of Ki-67 in Tumor Tissues
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Tumor tissues were sectioned and incubated with Ki-67 antibody (1/1000, ab279653, Abcam), followed by staining with horseradish peroxidase anti-rabbit IgG and diaminobenzidine. Subsequently, after incubation with mouse secondary antibody, EnVision G2 System/AP Rabbit Mouse Permanent Red (Dako, Glostrup, Denmark) was utilized to stain the sections, followed by counterstaining using hematoxylin. A fluorescent inverted microscope (Hitachi Limited, Tokyo, Japan) was utilized for observation.
4
CREBBP Immunohistochemical Staining
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The prepared tumor tissue sections were incubated with CREBBP antibody (1 : 1,000, ab2832) and stained with HRP-labeled anti-rabbit IgG or diaminobenzidine. Moreover, the sections were incubated with a mouse secondary antibody and stained with EnVision G2 System/AP Rabbit Mouse Permanent Red (Dako, Glostrup, Denmark). Finally, the sections were counterstained with hematoxylin and observed under a fluorescence inverted microscope (Hitachi Limited, Tokyo, Japan).
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