Fitc conjugated swine anti rabbit igs

The ink injected skin samples were cut out and fixed with 4% paraformaldehyde, embedded in polyester wax (VWR International), and sectioned for hematoxylin and eosin (H&E) staining observation. To detect Langerin⁺ cells, the back skin was removed at 24 hours after DT injection, and frozen skin specimens were sectioned and fixed by microwave irradiation and methanol. After blockade with 3% BSA, 2% skim milk, 0.1% Triton X100 in PBS, slides were stained with Langerin (Rabbit IgG poly; IMGENEX) antibody in 2% skim milk, 0.1% Triton X100 in PBS, followed by incubation with FITC-conjugated swine anti-rabbit Igs (DAKO) secondary antibody. Slides were counterstained with DAPI (Vector Laboratories).

The dual‐labeling immunofluorescence study was performed basically according to the method of the previous report (Han et al. 2003; Hashimoto et al. 2007; Saito et al. 2011). The following antibodies were used as the glomerular cell markers, mouse anti‐rat endothelial cell antigen‐1 (RECA‐1) antibody (Serotec, Oxford, UK), mouse monoclonal antibody to Thy1.1, a mesangial cell marker (mAb 1‐22‐3), mouse monoclonal antibody to synaptopodin, a podocyte marker (Progen, Heidelberg, Germany), mouse monoclonal antibody to nephrin, an SD marker (mAb 5‐1‐6), mouse monoclonal antibody to ZO‐1, an SD marker (Zymed Laboratories, San Francisco, CA), mouse monoclonal antibody to podocalyxin, a podocyte apical marker (Han et al. 2003), mouse monoclonal antibody to α3‐integrin, and a podocyte basal marker (Santa Cruz, CA). FITC‐conjugated swine anti‐rabbit Igs (DAKO) and tetramethylrhodamine isothiocyanate (TRITC)‐conjugated goat anti‐mouse IgG1 (Southern Biotechnology Associated) were used as secondary antibodies.

Immunohistochemical studies were performed basically according to the method previously reported (Kawachi et al. 2000; Ito et al. 2002; Han et al. 2003). A rabbit anti‐calcineurin pan A antibody was purchased from Millpore, MA, USA. The 3‐μm‐thick frozen sections were fixed with acetone for 1 min at room temperature, incubated at 4°C overnight with the anti‐CN‐A antibody, and stained with FITC‐conjugated swine anti‐rabbit Igs (DAKO, Glostrup, Denmark) at 37°C for 30 min. The anti‐CN‐A antibody recognized both CN‐A‐α and CN‐A‐β. The sections were observed with immunofluorescence microscopy (BX‐50; Olympus, Tokyo). The specificity of the anti‐CN‐A antibody was confirmed by preabsorption analysis with the GST‐fusion protein of CN‐A. To evaluate the CN‐A staining in the rat glomeruli, the staining was graded semiquantitatively as follows: continuous staining of >75% was score 4; 75–50% was score 3; 50–25% was score 2; and 25–0% was score 1. A score was assigned to each glomerulus, and 30 glomeruli of each rat were analyzed. The data are shown as mean ± SD of six rats (Nakatsue et al. 2005).