title: Comparative study for the detection of C4d in paraffin-embedded renal allograft biopsies by immunohistochemical techniques.
abstract: 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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Em-orn
authors.initials: EO
authors.lastname: Phanomsri
authors.forename: Nisana
authors.initials: N
authors.lastname: Namwat
authors.forename: Roongpet
authors.initials: R
authors.lastname: Tangrassameeprasert
authors.forename: Charn
authors.initials: C
authors.lastname: Charnteeraprawat
authors.forename: Anucha
authors.initials: A
authors.lastname: Paupiroj
mesh_terms: D001706:Biopsy; D002949:Citraconic Anhydrides; D015935:Complement C4b / Q000378:metabolism*; D019286:Endopeptidase K / Q000276:immunology / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006084:Graft Rejection / Q000175:diagnosis* / Q000276:immunology; D006801:Humans; D007124:Immunoenzyme Techniques; D016030:Kidney Transplantation / Q000276:immunology* / Q000473:pathology; D016612:Paraffin Embedding; D010446:Peptide Fragments / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*; D014184:Transplantation, Homologous / Q000276:immunology / Q000473:pathology
chemical_list: D002949:Citraconic Anhydrides; D010446:Peptide Fragments; D015935:Complement C4b; C032261:complement C4d; D019286:Endopeptidase K