Serum levels of IgM and IgG were determined by ELISA according to manufacturer’s instructions (Affymetrix/Invitrogen, Inc.). Immunoperoxidase, immunofluorescence, and Western blot of kidney tissues were performed as previously described.30 (link) Images for quantitative analysis were obtained on an Olympus BX51 microscope with an Olympus KP70 camera (Olympus America Inc.) and quantified using Image J (National Institute of Health). A transplant pathologist, blinded to study groups, counted the number of CD68+ cells within 20 glomeruli per allograft and determined the mean number of CD68+ cells per glomerulus. Immunoblot was performed on kidney lysates and reported as relative protein levels normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Antibodies directed at the following were used: CD68 (clone ED1, Biorad), GAPDH (clone 6C5, Abcam), C3 (55463, MP Biomedicals), C4d (12-5000, American Research Products), CD3 (ab5690, Abcam).
Protocol detail
Quantification of Kidney Allograft Immune Markers
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Allograft
Antibodies
Clone
Elisa
Glomerulus
Glyceraldehyde 3 phosphate dehydrogenase
Immunoblot
Immunofluorescence
Immunoperoxidase
Kidney
Microscope
Pathologist
Protein
Serum
Tissues
Transplant
Western blot
Corresponding Organization : University of Wisconsin–Madison
Other organizations : Wuhan University, Renmin Hospital of Wuhan University, Xiangya Hospital Central South University, Central South University
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Variable analysis
independent variables
- Treatment groups (not explicitly mentioned)
- Serum levels of IgM and IgG
- Number of CD68+ cells per glomerulus
- Relative protein levels of C3, C4d, and CD3 in kidney lysates
- Manufacturer's instructions for ELISA (Affymetrix/Invitrogen, Inc.)
- Immunoperoxidase, immunofluorescence, and Western blot methods as previously described
- Quantitative analysis using Olympus BX51 microscope and Image J software
- Blinded evaluation by a transplant pathologist
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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