Anti mouse igg

Sections of kidney tissues were incubated at 4 °C overnight with anti-mouse IgG (BioLegend, San Diego, CA, USA), anti-mouse C3 (Abcam, Cambridge, UK), or anti-mouse nephrin (Abcam, Cambridge, UK), washed, and incubated with anti-rat, anti-rabbit, or anti-mouse secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 594 for 2 h. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen/Thermo Fisher Scientific, San Diego, CA, USA). Control sections were treated in the same manner except that rat, rabbit, or mouse IgG were used in place of the primary antibodies. Confocal microscopy was performed and images were acquired using an LSM 800 confocal microscope (Zeiss, Oberkochen, Germany). Ten renal glomeruli areas were examined and averaged.

A custom multiplex assay was performed as previously described¹⁰⁹ (link)^,110 (link) in order to define the total levels and subclass profiles of HLA-A2-, HLA-A1- (NIH Tetramer Facility, HLA-A∗02:01 complexed with either CLGGLLTMV peptide from Epstein Bar Virus membrane protein or GLCTLVAML peptide from Epstein Bar Virus mRNA export factor ICP27, HLA-A∗01:01 complexed with VTEHDTLLY from cytomegalovirus pp50), and HSV-gD- (Immune Technology, USA) specific antibodies. Briefly, antigen-coupled microspheres were diluted in Assay Buffer (PBS + 0.1% BSA + 0.05% Tween20), and mAb or serum, followed by washing and detection with R-phycoerythrin (PE)-conjugated anti-Human IgG (Southern Biotech, USA), anti-mouse IgG (Biolegend, USA) or anti-human IgG1 (Southern Biotech, USA), IgG2 (Southern Biotech, USA), IgG3 (Southern Biotech, USA) and IgG4 (Southern Biotech, USA), respectively. These detection reagents have been characterized recently for specificity and sensitivity across IgG allotypes.¹¹¹ (link) Median fluorescent intensities (MFI) were acquired on a FlexMap 3D (Luminex, USA).

HCFs were cultured on 4-well cell culture slides (30,104; SPL Life Sciences, Pocheon, Korea). After fixation in 4% paraformaldehyde, the cells were incubated with 0.1% Triton X-100 in PBS for 30 min, blocked for 1 h with 1% bovine serum albumin in PBS, and incubated with rabbit anti-KLF4 (Abcam, Cambridge, UK) and rabbit anti- α-SMA (Sigma Chemical Co, MO, USA) antibodies. The secondary antibodies were anti-rabbit IgG and anti-mouse IgG (BioLegend, CA, USA). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma Chemical Co). Fluorescence images were obtained using an Olympus CKX41 microscope (Tokyo, Japan).