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Optical Clearing and Imaging of Immunolabeled Kidney Tissue
Published on: July 22, 2019
Comparative study for the detection of C4d in paraffin-embedded renal allograft biopsies by immunohistochemical
Em-orn Phanomsri1, Nisana Namwat, Roongpet Tangrassameeprasert
1Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
Insights
This study compared indirect immunofluorescence (IF) and immunoperoxidase (IP) methods for detecting C4d deposition in kidney allografts. Citraconic anhydride unmasking enhanced C4d staining signals for both IF and IP techniques, aiding rejection diagnosis.
Area of Science:
- Nephrology
- Immunology
- Histopathology
Background:
- Peritubular capillary C4d deposition is a diagnostic criterion for acute humoral rejection in kidney allografts.
- Effective staining techniques for C4d protein are crucial but not well-established.
Purpose of the Study:
- To compare C4d staining results using indirect immunofluorescence (IF) and immunoperoxidase (IP) methods.
- To evaluate antigen unmasking procedures for formalin-fixed, paraffin-embedded renal allograft tissue.
Main Methods:
- C4d protein detection in renal allografts using IF and IP.
- Comparison of antigen unmasking techniques: (i) citraconic anhydride with proteinase K, (ii) citrate buffer microwave heating, (iii) proteinase K digestion.
Main Results:
- 0.05% citraconic anhydride with water bath heating enhanced IP signals.
- 0.05% citraconic anhydride with water bath heating plus proteinase K digestion greatly enhanced IF signals.
- Prevalence of C4d staining in peritubular capillaries was 68% (17/25), with similar results between methods.
Conclusions:
- 0.05% citraconic anhydride treatment is effective for unmasking C4d deposition.
- This unmasking technique is beneficial for both IP and IF methods in renal allograft analysis.
Background:
Peritubular capillary complement C4d deposition is one of the criteria for diagnosis of acute humoral rejection in kidney allografts. Little information is provided for the effective technique to stain C4d protein.
Objective:
To compare C4d staining results by using indirect immunofluorescence detection (IF) with immunoperoxidase methods (IP) in formalin-fixed, paraffin-embedded renal allograft tissue.
Material And Method:
C4d protein was detected in renal allograft tissues by IF and IP methods. The antigen unmarking procedures were used including (i) heating with 0.05% citraconic anhydride in a water bath plus proteinase K digestion, (ii) heating with 10 mM citrate buffer in a microwave and (iii) digesting with proteinase K for comparing the deposition of C4d in paraffin-embedded tissues.
Results:
The results showed that the unmarking solution containing 0.05% citraconic anhydride pH 7.4 and heating in a water bath revealed a signal enhancement in the IP method whereas the solution containing 0.05% citraconic anhydride pH 7.4 and heating in a water bath plus proteinase K digestion showed a greatly enhanced signal in the IF method. The prevalence of C4d staining detected in peritubular capillaries was 68% (17/25) and the results observed for both methods were similar.
Conclusion:
0.05% citraconic anhydride in a water bath with or without proteinase K digestion is useful for unmasking C4d deposition in peritubular capillaries of renal allografts performed by IP and IF methods.