Figure 2.

Staining of C5b-9 in a healthy and a diseased kidney. Examples of staining of C5b-9 from our laboratory are shown. (A) In a healthy kidney, staining was present in the vascular pole of the glomerulus and the vascular wall of extraglomerular arteries and focally with less intensity along Bowman’s membrane and the tubular basement membrane. This tissue was obtained with a biopsy from a living donor before kidney transplantation. (B) In a kidney of a patient with aHUS, staining was present along the glomerular capillary wall, in the vascular wall of extraglomerular arteries and focally along Bowman’s membrane and the tubular basement membrane. This tissue was obtained with a clinically indicated biopsy. Both tissues were fixed, paraffin-embedded, and sectioned. After deparaffinization (xylol and ethanol) and antigen retrieval (PBS-0.1% Proteinase XXIV, P8038, Sigma), sections were washed and endogenous peroxidase was blocked (PBS, 0.1% NaN₃, 1% H₂O₂) for 30 min at room temperature. Sections were washed (PBS) and incubated with mouse anti-human C5b-9 (2 µg/ml, aE11, HM2167, Hycult Biotech, Uden, the Netherlands) or an isotype control (mouse IgG2a, 2 µg/ml, X0943, Dako, Jena, Germany) in PBS with 1% BSA over night at room temperature. Next day, slides were washed and incubated with goat anti-mouse horseradish peroxidase (HRP, 5 µg/ml, P0447, Dako) for 30 min at room temperature. Slides were washed and incubated with rabbit anti-goat HRP (2.5 µg/ml, P0449, Dako) for 30 min at room temperature. Slides were washed and developed using NovaRED following protocol (Vector Labs, Peterborough, UK) and counterstained (Mayer’s hematoxylin, 1.09249.0500, Merck, Darmstadt, Germany) for 25 s. Slides were not counterstained with eosin, which explains why tubules may seem dilated. Slides were dried overnight at room temperature before being covered using entellan (1.07961, Merck).