Consecutive 4-μm thick sections were immunohistochemically stained using the immunoperoxidase technique described previously [45 (link)], with the primary antibodies listed in Table 5 and appropriate secondary antibodies (Medical and Biological Laboratories, Nagoya, Japan) (all 0.2 µg/mL). The tissue sections were then color-developed with diamine benzidine hydrochloride (Dako, Glostrup, Denmark) and counterstained with Meyer’s hematoxylin (Sigma-Aldrich Chemical Co., St. Louis, MO, USA). Staining indices were evaluated by examining 1000 epithelial cells, and the frequency of positive nuclear staining was determined. For p53 and NS, an index of more than 10% was considered to represent a “positive case”. TERT evaluation was performed by assessing 100 cells at the bottom of the glands, and a positive cell number per 20 cells was considered to represent TERT expression.
Appropriate secondary antibodies
Manufactured by Medical & Biological Laboratories
Sourced in Japan
Appropriate secondary antibodies are laboratory reagents used to detect and visualize target proteins in samples. They are designed to bind to the primary antibodies specific to the target proteins, allowing for their indirect detection and quantification. These secondary antibodies are labeled with various reporter molecules, such as fluorescent dyes or enzymes, to enable signal detection and amplification. They play a crucial role in immunoassays, immunohistochemistry, and other protein analysis techniques.
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3 protocols using appropriate secondary antibodies
1
Immunohistochemical Quantification of Biomarkers
2
Immunohistochemical Analysis of CKB and MTCK
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Tissue microarray slides were processed for the immunohistochemical analysis of CKB and MTCK. Tissue micro array slides were incubated with antibodies against CKB (0.2 μg/mL, ab151579, Abcam, Cambridge, MA, USA) and MTCK1 (0.2 μg/mL, 89-7263-83, Abbexa Ltd., Cambridge, UK) and appropriate secondary antibodies (Medical and Biological Laboratories, Nagoya, Japan) (all 0.2 μg/mL). The tissue sections were then color-developed with diamine benzidine hydrochloride (Dako, Glostrup, Denmark) and counterstained with Meyer’s hematoxylin (Sigma-Aldrich Chemical Co., St. Louis, MO, USA). To evaluate CKB and MTCK, we counted cells that exhibited immunoreactivity in the cytoplasm and scored staining strength as grade 0–3. The product of the grade (0–3) and frequency of positive cells (0–1) is the expression index.
3
Immunohistochemical Evaluation of Colorectal Biomarkers
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Consecutive 4-μm sections were immunohistochemically stained using the immunoperoxidase technique described previ-ously [13] , with primary anti-CD47 (Abcam, Cambridge, UK), anti-CD44 (Santa-Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-CK20 (DAKO Corp, Glostrup, Denmark), antivimentin (DAKO), anti-programmed cell death ligand (PD-L) 1, antihuman mutL homolog (hMLH)-1 (Abcam), and appropriate secondary antibodies (Medical and Biological Laboratories, Nagoya, Japan) (all 0.2 µg/mL). The tissue sections were then color-developed with diamine benzidine hydrochloride (DAKO, Glostrup, Denmark) and counterstained with Meyer's hematoxylin (Sigma). We counted cells that exhibited immunoreactivity at the cytoplasmic membrane and scored staining strength between 0 to 3 (where a score of 1 was used to describe the expression level in a normal colonic epithelium). The staining index was then calculated as the staining strength score multiplied by the staining area (%).
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