Lax s software

OCT-embedded cryo kidney sections (4 μm) were fixed in acetone for 20 min and blocked with 10% BSA for 1 h. Sections were then incubated with rabbit anti-mouse C3 (Cat: PA1-40,288), APC conjugated anti-mouse CD4 (Cat: 17–0042-82), Alexa-488 conjugated anti-sheep IgG (Cat: A11015), or Alexa-488 conjugated anti-mouse IgG (Cat: A21202), for 1 h at room temperature, followed by Rhodamine-conjugated anti-rabbit (Cell Signaling Technology Beverly, MA. Cat: R6394, 1:100 dilution) for C3 secondary antibodies staining for 1 h. All primary Abs were purchased from Life Technologies (Carlsbad, CA, 1:100 dilution). Slides were covered with Fluoromount-G mounting medium (SouthernBiotech, Birmingham, AL) after three times washing with PBS. Images were acquired with a Leica DMi8 fluorescent microscope (Buffalo Grove, IL) and analyzed with the LAX S software (Leica Microsystems Inc.). CD4⁺ T cell numbers were counted in four (200 ×) low-power fields in each section. Sections from at least 4 mice were counted and analyzed.

Spleen sections (4 μm) were fixed in acetone for 10 min and then blocked with 5% BSA in TBS buffer with 0.1% Tween for 20 min. Sections were then incubated with 1:100 dilutions of anti-mouse antibodies (IgD and GL-7) from BD Biosciences (San Jose, CA) and (anti-Ezh2, anti-rabbit IgG-Alex 488, anti-rabbit IgG-Rhodamine red) from Cell Signaling Technology (Beverly, MA). Images were acquired using a Leica DMi8 fluorescence microscope (Buffalo Grove, IL) and analyzed with the LAX S software developed by Leica Microsystems Inc.