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Original ArticleOpen Access
Comparative Study for the Detection of C4d in Paraffin-Embedded Renal Allograft Biopsies by Immunohistochemical Techniques
Phanomsri E 
,
Namwat N ,
Tangrassameeprasert R ,
Charnteeraprawat C ,
Paupiroj A
,
Namwat N ,
Tangrassameeprasert R ,
Charnteeraprawat C ,
Paupiroj A 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 10mM 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 pH7.4 and heating in a water
bath revealed a signal enhancement in the IP method whereas the solution containing 0.05% citraconic anhydride pH7.4 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.
Keywords: Antigen unmarking, Citraconic anhydride, Protease K, Allograft
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 10mM 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 pH7.4 and heating in a water
bath revealed a signal enhancement in the IP method whereas the solution containing 0.05% citraconic anhydride pH7.4 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.
Keywords: Antigen unmarking, Citraconic anhydride, Protease K, Allograft
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