Fitc conjugated anti human igg

Cells were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA (Nacalai Tesque, Inc.). After washing with PBS, the cells were treated with LpMab-7 and chLpMab-7 (1 μg/ml) for 30 min at 4°C followed by incubation with Oregon green-conjugated anti-mouse IgG or FITC-conjugated anti-human IgG (Thermo Fisher Scientific Inc.). Fluorescence data were collected using a Cell Analyzer EC800 (Sony Corp., Tokyo, Japan).

Mouse kidneys were fixed in 10% neutral formalin, dehydrated, and embedded with paraffin. Tissue blocks were sectioned (2 µm) and followed by periodic acid–Schiff (PAS) staining. Histopathological evaluation was performed on a scale of 0–3 according to cell proliferation and cell infiltration by two observers blind to the protocol as previously described (37 (link)).
To assess immune deposition in the glomeruli, kidney samples were collected and embedded in optimal cutting temperature (OCT) compound (SAKURA). Renal blocks were sectioned (4 μm), and slides were fixed in pre-cold acetone for 10 min. Tissue slides were then washed with PBS twice and incubated with FITC-conjugated antihuman IgG (Thermo Fisher, #A11001) and FITC-conjugated antihuman complement component 3 (C3) antibodies (Thermo Fisher, #PA1-28933) at 4°C overnight. Sections were washed and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) in mounting medium (Vector Laboratories). Fluorescence signals were examined using a fluorescence microscopy (Olympus).

The internalization assay was carried out as previously described [34 (link)]. Briefly, the cells were incubated with cycloheximide (75 µg/mL) to prevent the transport of de novo synthesized c-Kit to the cytoplasmic membrane. Then, the cells were blocked with human BD Fc block™ (BD Biosciences, San Diego, CA, USA), to inhibit the Fc receptor-mediated internalization of antibodies, and 5% BSA (CellNest, Gyeonggi-do, Korea) at RT for 10 min. The cells were washed with PBS and incubated in the presence or absence of the 4C9 antibody (1 µg/mL) at 4 °C or 37 °C for 1–4 h. The cells were then incubated with FITC-conjugated anti-human IgG (0.3 μg/mL, Invitrogen) for 1 h, washed three times with PBS, and analyzed using a CyFlow^® Cube 6 (PARTEC).