 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 (Complement 4d) Antibody |
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Clone: 10-11, Mouse anti-Human |
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Supplier: Biogenesis |
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Catalog Number: 2222-8004 |
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Dilution: 1:500 - 1:600 |
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Incubation Time/Temp: 60min/room temperature |
Antigen Retrieval
| Device: Steamer |
| Buffer/pH value: EDTA/pH 8.0. It has been observed that pretreatment with 88% formic acid for 20 minutes at room temperature is also beneficial. |
| 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 of the glomeruli and in some cases the peritubular capillary |
| 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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There is no need to perform antigen retrieval for acetone fixed frozen sections |
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