Alexa fluor 594 labeled donkey anti mouse igg

We performed IF staining of renal biopsy specimens and tonsillar specimens obtained from patients who underwent tonsillectomy after having been diagnosed with IgAN and in whom both kidney and tonsillar specimens were available. Indirect double IF staining with Cnm antiserum and a mouse monoclonal antibody against human CD68, a marker for macrophages (PG-M1, Agilent), was performed using the following secondary antibodies: Alexa Fluor 488-labeled donkey anti-rabbit immunoglobulin G (IgG; Molecular Probes, Eugene, OR) and Alexa Fluor 594-labeled donkey anti-mouse IgG (Molecular Probes).
IF staining for Gd-IgA1 in kidney tissues from IgAN patients was performed using KM55, a Gd-IgA1- specific monoclonal antibody that has specific immunoreactivity for glomerular IgA deposition and can thus reflect the pathogenesis of IgAN^{49 (link)}. Gd-IgA1 staining has been reported to be useful for diagnosis of IgAN^{50 (link)} and is hence performed with biopsy samples at many institutions, including ours, for such diagnosis. The Alexa Fluor 488-labeled donkey anti-rabbit IgG (Molecular Probes) secondary antibody was used for detection. Based on the degree of staining, IgAN patients were divided into two groups: (i) strong (1+) and (ii) weak (±) (there was no patient of negative (−) staining). We calculated the percentage of the number of patients in (i) to the number of ones in the IgAN group.

After 24 hours of treatment with Mn, cultured slices were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer containing 4% sucrose for 2 h and were frozen subsequently. After the sections were rinsed with PBS, they were permeabilized and blocked in 0.5% Triton X-100 in PBS containing 5% donkey serum (Jackson ImmunoResearch Laboratories Inc., USA), then they were incubated with these primary antibodies overnight at 4°C: rabbit anti-C-terminal alpha-synuclein polyclonal antibody (1:50) and mouse anti-calpain 1 polyclonal antibody (1:100). After being rinsed with PBS, slices were incubated with these secondary antibodies for 2 h at room temperature: Alexa Fluor 488-labeled donkey anti-rabbit IgG and Alexa Fluor 594-labeled donkey anti-mouse IgG (1:1000; Molecular Probes, Invitrogen, Carlsbad, CA). The fluorescent signal was examined on an Olympus confocal microscope (FV 1000S- IX81, Olympus, Japan), using the 40× objective lens. Confocal laser scanning microscope digital images were collected and saved in TIFF format, using FV10-ASW software (ver. 03.00.01.15, Olympus).

Cells were seeded on glass coverslips, and were infected with EV71 at the indicated time, followed by fixation with 4% paraformaldehyde for 15 min at room temperature. After being washed twice with PBS, the cells were treated with specific primary antibodies against the proteins. The respective secondary antibody was Alexa Fluor 594‐labeled donkey anti‐mouse IgG (Molecular Probes, Life technologies) diluted at 1 : 1000. Images were acquired with an Olympus Fluoview FV10i laser scanning confocal microscope (Tokyo, Japan).