Alexa fluor 568 labeled donkey anti goat igg

Kidneys were embedded in OCT compound and frozen at −70 °C. Samples were cut into 5-μm-thick sections, fixed in 4% paraformaldehyde, and washed 3 times in cold PBS. Nonspecific binding was blocked with 1% normal goat serum in PBS with Tween 20 for 30 min. Sections were incubated with goat anti-mouse IgG (1:100; Serotec, Oxford, UK) and rabbit anti-mouse complement 3 (C3) (1:100; Abcam) at 4 °C overnight, washed 3 times with PBS, incubated with Alexa Fluor 568–labeled donkey anti-goat IgG and Alexa Fluor 488-labelled donkey anti-rabbit IgG (each 1:100; Invitrogen) at room temperature for 1 h, and washed 3 times with PBS. Sections were mounted in a mounting solution (Vector Laboratories, Burlingame, CA, USA) and examined under a laser scanning confocal microscope (LSM 780; Carl Zeiss, Oberkochen, Germany). Two pathologists independently assigned semi-quantitative scores indicating the intensity and distribution of immunofluorescence staining for IgG and C3, on a scale ranging 0–3, in which 0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining. The average score obtained by the 2 pathologists was determined.