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Updated: Jul 9, 2026
Whole-Kidney Three-Dimensional Staining with CUBIC
Published on: July 18, 2022
C4d Immunohistochemistry in glomerulonephritis with different antibodies
Taisei Suzuki1, Shigeru Horita2, Koji Kadoya3
1Molecular Pathology, Biomolecular and Integrated Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennohdai, Tsukuba, Ibaraki, 305-8575, Japan.
Insights
This study found that anti-C4d polyclonal antibodies (C4dpAb) show intrinsic C4d deposition in normal glomeruli via immunofluorescence (IF), unlike paraffin immunoperoxidase (IP) staining. This highlights C4d
Area of Science:
- Nephrology
- Immunology
- Pathology
Background:
- C4d deposition in kidneys is crucial for diagnosing antibody-mediated rejection in renal transplants.
- Intrinsic C4d deposition in normal glomeruli is observed with immunofluorescence (IF) using anti-C4d monoclonal antibodies (mAbs), but its role is unknown.
- Anti-C4d polyclonal antibodies (C4dpAb) for paraffin immunoperoxidase (IP) staining are less utilized and have not clearly shown intrinsic C4d.
Purpose of the Study:
- To establish a stable and reproducible method for C4d detection using C4dpAb.
- To characterize the staining properties of C4dpAb.
- To compare C4dpAb staining with IF using mAbs in kidney tissue.
Main Methods:
- Compared C4dpAb with mAb in adjacent sections of human diseased kidneys using IF.
- Compared IP staining with IF staining for C4dpAb.
- Investigated two antigen retrieval methods for IP staining.
Main Results:
- C4dpAb showed similar glomerular staining patterns and intensity to mAbs via IF, revealing intrinsic C4d.
- Intrinsic C4d in normal glomeruli was largely undetectable by IP with C4dpAb, while IF showed distinct staining.
- C4dpAb IP staining sometimes appeared weaker than IF, suggesting non-intrinsic C4d deposition indicative of complement activation.
Conclusions:
- C4dpAb is valuable for immunohistochemistry, prompting a re-evaluation of C4d's role in glomerular diseases.
- The study established a reproducible C4dpAb staining protocol.
Background:
The presence of C4d in the kidney is generally detected particularly for the diagnosis of antibody-mediated rejection in renal transplants. In frozen sections of immunofluorescence (IF) staining with anti-C4d monoclonal antibodies (mAbs), we noted intrinsic C4d deposition even in normal glomeruli though their pathogenic or an intrinsic role is unkown. An anti-C4d polyclonal antibody (C4dpAb), which is suitable for paraffin immunoperoxidase (IP) staining, is less used than mAbs, and it has demonstrated that intrinsic C4d is not evident. To establish a stable and reproducible procedure for C4d detection with the C4dpAb and to determine the staining characteristics of it, the present study aimed to test whether the method was comparable with IF with a mAb.
Methods:
We compared the C4dpAb with the mAb in adjacent sections of human diseased kidneys, and then compared IP with IF of C4dpAb. Two ways of antigen retrieval was examined for IP.
Results:
On comparing the two antibodies for glomerular staining with IF, we found that the pattern and intensity (C4dpAb showed intrinsic C4d with IF) were similar. In addition, C4dpAb staining with IP and IF demonstrated that the intrinsic staining in the normal glomerulus was mostly undetectable by IP, whereas IF showed distinct staining. Likewise, C4d deposition with IP in some cases was apparently weaker than that on IF, suggesting that this deposition is not intrinsic but indicates pathogenic complement activation.
Conclusions:
The advantage of the C4dpAb for immunohistochemistry is of value for reconsidering the role of C4d in glomerular diseases.
