Efluor 570

Renal immune complex deposition levels were observed using immunohistochemistry (IHC). Following deparaffinization, rehydration, and antigen retrieval, the slides were blocked with 2.5% normal horse serum, washed, and incubated with ImmPRESS Anti-Mouse IgG (Vector MP-7802). Antibody binding was visualized by incubating slides in a DAB working solution (Vector MP-7802). The slides were imaged using an Olympus BX51 light Microscope (Tokyo, Japan). The staining levels (arbitrary gray units) of 30 glomeruli per slide were quantified using ImageJ (Version 1.52a, NIH, USA). A second series of slides, for illustration purposes only, were counterstained with hematoxylin and incubated in bluing reagent.
Renal macrophage infiltration levels were evaluated through immunofluorescence (IF). Kidney sections were blocked by 10% (v/v) goat serum, followed by incubation under rat anti-mouse F4/80 monoclonal antibody eFluor 570 (eBioscience; 41–4801-82;). Subsequently, coverslips were mounted with Prolong Antifade Mountant (Life Technologies, USA). The macrophage infiltration levels were assessed by counting positively stained macrophages from 50 glomeruli per slide under an Olympus BX51 Fluorescence Motorized Microscope.

Renal immune complex deposition was evaluated by immunohistochemistry (IHC), as described previously.^{21 (link)} The slides were visualized using an Olympus BX51 light microscope (Tokyo, Japan). Thirty glomeruli per slide were analyzed for the staining levels (arbitrary gray units) using ImageJ software (Version 1.52, NIH).
Renal macrophage infiltration levels were assessed by immunofluorescent staining (IF).^{21 (link)} In brief, kidney slides were blocked and then incubated with rat anti-mouse F4/80 antibody eFluor 570 (eBioscience). Thirty glomeruli per slide were investigated to count positively stained macrophages.