Four serial sections of 5 μm per paraffin block are obtained for the following immunohistochemistry staining. These sections were first baked at 60 °C and then deparaffinized in xylene and ethanol. After hydration, 3% hydrogen peroxidase was utilized to block endogenous peroxidase activity. Standard antigen retrieval was conducted via heating the sections immersed in citric acid solution (pH = 6.0) in a pressure boiler. Subsequently, these slides were incubated with the primary antibodies [CD20 (60271-1-Ig, Proteintech, 1:5000); CD4(ab133616, Abcam, 1:500), CD8(ab85792, abcam, 1:400), CD68(ab959, Abcam, 1:6000)] at 4°Covernight, and then incubated with second antibody. After 3,3'-diaminobenzidine tetrahydrochloride staining and hematoxylin counterstaining, the slides were scanned for further quantitative analysis. The density of CD4 + , CD20,CD68 and CD8 + T cells both invasive margin (IM) and in the core of the tumor (CT) were automatically calculated using ImageJ software (version 1.48). The software generally contains positive cells and a positive nucleus, and we used its ratio (positive cells/positive nucleus) to represent the expression status of four immune cells in CRC tissues.
Ab959
Manufactured by Abcam
Ab959 is a lab equipment product from Abcam. It is a mono-specific antibody that binds to a specific target protein. The core function of this product is to facilitate detection and quantification of the target protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using ab959
1
Immunohistochemical Quantification of Immune Cells in CRC
2
Immunohistochemical Quantification of Immune Cell Markers
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Four serial sections of 5 μm per paraffin block for subsequent immunohistochemistry staining were obtained. Initially, these sections were subjected to baking at 60 °C, followed by deparaffinization in xylene and ethanol. After hydration, we used 3 per cent hydrogen peroxidase to block endogenous peroxidase activity. For standard antigen retrieval, the sections were heated in a citric acid solution (pH = 6.0) using a pressure boiler. Subsequently, the slides were incubated with primary antibodies [CD4 (ab133616, Abcam, 1:500); CD8 (ab85792, Abcam, 1:400); CD20 (60271-1-Ig, Proteintech, 1:5000); CD68 (ab959, Abcam, 1:6000)] overnight at 4 °C, followed by incubation with a secondary antibody. After staining with 3,3ʹ-diaminobenzidine tetrahydrochloride and counterstaining with haematoxylin, the slides were scanned for further quantitative analysis. The density of CD4+, CD8+, CD20 and CD68 T cells at the invasive margin (IM) was automatically calculated using ImageJ software (version 1.48).
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