Donkey anti rabbit igg

For CLSM analysis, 0.5 × 10⁶ ds Red A. fumigatus conidia were applied to the apical surface and at several time points post infection as indicated in the Figure legends or Results, inocula were removed by gentle suction and the well was fixed using 4% paraformaldehyde solution for 10 minutes. After fixation the cells were treated as described above except staining the slides for mucin using rabbit anti-human MUC5B antibody (1:80; Abcam), followed by donkey anti-rabbit IgG (1:50 Biolegend). Again, actin localized to tight junctions was identified by using phalloidin-650 (1:20; Cell Signaling Technology). Cilia were stained using an anti-tubulin antibody (1:10; BD Pharmingen), and nuclei using Hoechst 33342 (Cell Signaling Technology). Images were captured using a Leica SP5 confocal laser scanning microscope and analysed using the LAS AF Lite or Imaris software. For each condition at least 50 cells were analyzed and used for quantifications.

The medium used throughout was RPMI 1640 medium (Sigma-Aldrich. Inc., St. Louis, MO, USA) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 µg/ml streptomycin (hereinafter called RPMI medium). Mouse monoclonal antibodies (mAbs) specific for HTLV-I Tax (clone Lt-4), OX40 (clone B-7B5), and KLH (IgG3, clone KLH-3, Tanaka et al., unpublished) were purified in our laboratory from ascites fluids of CB.17-SCID mice carrying the appropriate hybridomas. The ascites fluid samples were subjected to ammonium sulfate precipitation followed by gel filtration with Superdex G-200 (GE Healthcare, Tokyo, Japan). mAbs were labeled with either fluorescein isothiocyanate (FITC) or HiLyte Fluor™ 647 using commercial labeling kits (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. FITC-, PE- or PE-Cy7-labeled mouse mAbs against human CD3, CD4, CD8, CD14, CD19, CD56, and CD83 (clone H15e), and FITC-labeled goat anti-rat IgG, donkey anti-rabbit IgG, and HRP-labeled goat anti-rat IgG antibodies were purchased from BioLegend (Tokyo, Japan). Rabbit polyclonal IgG anti-rat CD83 antibody was obtained from Sino Biological (Beijing, China). Prostaglandin E2 (PGE2) was purchased from Sigma-Aldrich. Inc., (St. Louis, MO, USA).

Cells grown on coverslips were first subjected to fixation and permeabilization with BD Cytofix/Cytoperm^TM Kit. Then, samples were incubated with primary antibodies against TUBB3 and glial fibrillary acidic protein (GFAP, Poly28400, BioLegend), followed by fluorescence-conjugated secondary antibodies of rat anti-mouse IgG_2a (BioLegend) and donkey anti-rabbit IgG (BioLegend). Counterstain for the nuclei was performed using diamidino-2-phenylindole (DAPI, Sigma-Aldrich). After washing, specimens were mounted with Dako fluorescence mounting medium (Agilent, Santa Clara, USA) and examined under a Leica confocal microscope.