 Methods & Techniques For Histologists and Immunohistochemists |
C4d Antibody Staining Protocol for Immunohistochemistry
Description: Deposition of C4d, a split product of C4 of the classical pathway of complement, in peritubular capillaries (PTCs) has been shown to be a sensitive marker for antibody-mediated (humoral) rejection in renal transplant biopsies. Some studies also suggest that C4d in PTCs is specific for humoral rejection or, at least, for the presence of donor-specific antibodies. The capillary C4d deposition can also be used to diagnose antibody-mediated injury due to non-HLA antidonor antibodies, as shown by a report from Japan analyzing rejection patterns in ABO-incompatible renal transplantation.
Primary Antibody
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Name: C4d Antibody |
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Clone: Polyclonal, Rabbit anti-Human |
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Supplier: Biomedica |
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Catalog Number: BI-RC4D |
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Dilution: 1:25 - 1:50 |
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Incubation Time/Temp: 60min/room temperature |
Antigen Retrieval
| Device: Steamer |
| Buffer/pH value: Citrate buffer/pH 6.0 or EDTA/pH 8.0 |
| Heat/Cool Temperature: 95-100 ºC/room temperature |
| Heat/Cool Time: 20 minutes/20 minutes |
Detection Methods
| Standard Method: ABC Method or LSAB Method |
| Enhanced Method: Polymeric Methods |
Chromogen Substrate
| Reagent: DAB |
| Incubation Time/Temperature: 1-3 minutes/room temperature |
Counterstain
| Reagent: Gill's Hematoxylin or Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| Staining Pattern: Cytoplasmic |
| Images: Search image |
Additional Information:
| Tissue Type: Kidney |
| Fixation: Formalin-fixed paraffin sections, or acetone fixed frozen sections |
| Positive Control: Kidney |
| Negative Control: Omit primary antibody, isotype control, absorption control |
| Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary |
Notes:
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1. EDTA buffer pretreatment produces stronger staining intensity than citrate buffer. 2. Overnight incubation increases staining intensity. 3. More sensitive detection system is required to produce optimal result. Polymeric method such as Envision+, ImmPress is recommended. 4. For frozen sections, a higher dilution of primary antibody may be used. |
References:
1.
Bruno Watschinger
and Manuel Pascual
(2002) Capillary C4d Deposition as a Marker of Humoral Immunity in
Renal Allograft Rejection.
J Am Soc Nephrol 13:2420-2423
2. Collins AB, Schneeberger E, Pascual M, Saidman S, Williams W, Tolkoff-Rubin N, Cosimi AB, Colvin RB (1999) Complement activation in acute humoral renal allograft rejection: Diagnostic significance of C4d deposits in peritubular capillaries. J Am Soc Nephrol 10: 2208–2214
3. Bohmig GA, Exner M, Habicht A, Schillinger M, Lang U, Kletzmayr J, Saemann MD, Horl WH, Watschinger B, Regele H (2002) Capillary C4d deposition in kidney allografts: A specific marker of alloantibody-dependent graft injury. J Am Soc Nephrol 13: 1091–1009
4. Regele H, Böhmig GA, Habicht A, Gollowitzer D, Schillinger M, Rockenschaub S, Watschinger B, Kerjaschki D, Exner M (2002) Capillary deposition of complement split product C4d in renal allografts is associated with basement membrane injury in peritubular and glomerular capillaries: A Contribution of humoral immunity to chronic allograft rejection. J Am Soc Nephrol 13: 2371–2380