Fluorescein 5 isothiocyanate fitc

bPEI was fluorescently labeled with fluorescein-5-isothiocyanate (FITC) (Thermo Fisher). 15 mg of bPEI was dissolved in 0.1 M NaHCO₃ (pH = 9) at 2.5 mg/mL, and 2 mg of FITC was dissolved in 200 µL of DMSO. The FITC solution was added drop-wise to the bPEI solution and allowed to stir for 3 h at RT. Free FITC was removed using 10,000 MWCO centrifugal filters, and bPEI-FITC was stored in water. The bPEI concentration was determined by a TNBS assay as described above, and the presence of FITC showed no interference with the results, as reported before [20 (link)]. For the conjugates, 0.2 mg of FITC (1 mg/mL) in DMSO was added dropwise into 2 mg of HAI-bPEI synthesized via the sulfo-SMCC approach and allowed to react for 3 h with stirring. The concentration of bPEI in the conjugate was determined by a TNBS assay with a bPEI-FITC standard curve. Holo human Transferrin was fluorescently labeled with Texas Red modified with succinimidyl ester (Thermo Fisher) following the manufacturer’s protocol. Amine modified siRNA was fluorescently labeled with Alexa Fluor 488 (Thermo Fisher) following the manufacturer’s protocol, followed by purification using ethanol precipitation and silica spin column binding as reported before [31 (link)].

The tissue material collected from the kidney biopsy was rapidly frozen in liquid nitrogen and cut to a thickness of 5 µm using a cryostat (Thermo Scientific, Cheshire, UK). Biopsy slides were fixed in a cold 1:1 mixture of alcohol and acetone at 4 °C for 5 min. The slides were then dried at room temperature for 5 min and washed three times using phosphate-buffered saline (PBS, pH 7.4; Thermo Fisher Scientific, Waltham, MA, USA). The samples were incubated with antibodies against IgA, IgG, and IgM, as well as C3 and C1q, which are complement proteins. They were then labeled with fluorescein 5-isothiocyanate (FITC; Thermo Fisher Scientific, Waltham, MA, USA) in the dark at room temperature. A 1:20 dilution of antibodies was used. Following incubation, the slides were washed three times in PBS and embedded in a glycerol medium. The presence, composition, and location of immunoglobulins and complement protein deposits were assessed using an Olympus BX43 research microscope with an EPI fluorescence kit (Olympus, Tokyo, Japan).

α-Lac from bovine milk-type III-calcium depleted (L 6010), (3-(4,5-dimethylthiazol-2-yl)-2,5-dephenyltetrazolium bromide (MTT), dialysis membranes (cut-off, 12000 Da), acridine orange, Ludox®, penicillin, and tris(hydroxymethyl) aminomethane (Tris) were obtained from Sigma-Aldrich. Fetal bovine serum (FBS), RPMI 1640 culture medium, and trypsin were purchased from GIBCO. DOX-HCl, ethidium bromide, ethylenediaminetetraacetic acid (EDTA), and dimethyl sulfoxide (DMSO) were from Merck. Fluorescein 5-isothiocyanate (FITC) and PTX were obtained from Thermo-Fisher Scientific (Waltham, MA) and Stragen Pharma, respectively. Flasks, petri dishes, and 96-well plates were purchased from Nunc (Denmark).