Affinipure donkey anti rabbit igg h l

We obtained anti-human CCR5 45531 from R and D Systems (Minneapolis, MN), anti-actin from Sigma Aldrich (St. Louis, MO), anti-Giantin 924302 from Biolegend (San Diego, CA), Alexa Fluor 488 FluoroNanogold goat anti-mouse IgG Fab from Nanoprobes.com (Yaphank, NY), AffiniPure Donkey Anti-Rabbit IgG (H + L) from Jackson ImmunoResearch (West Grove, PA), Alexa Fluor 488 goat Anti-Mouse IgG (H + L) from Invitrogen (Carlsbad, CA), Anti-SNAP-tag Antibody (Polyclonal) from New England Biolabs (Ipswichm, MA). Hybridoma cells expressing anti-Myc 9E10 were obtained from ATCC. Saponin (from quillaja bark), LPS (from Escherichia coli 0111:B4), glutaraldehyde, and fumonisin B1 (from Fusarium moniliforme) was purchased from Sigma Aldrich (St. Louis, MO). Recombinant human CCL5 was obtained from R and D Systems (Minneapolis, MN).

Cells were grown on coverslips in 24-well plates. When cells reached 80–90% confluence, medium was aspirated and cells were washed with 500 μl PBS. Cells were fixed in 4% formaldehyde for 10 min and washed as described above. The cells were permeabilized by 0.1% Triton X-100 in PBS for 3 min at room temperature. After washing with PBS, the cells were blocked for 1 h in PBS containing 5% donkey serum.Then, 30 µl anti-KOR antibody (1:100, Abcam, UK) was added to each coverslip, which were incubated overnight at 4 °C in the dark. After washing, coverslips were stained with Acti-stain 488 phalloidin (Cytoskeleton, USA, #PHDG1) and Dylight 594-conjugated AffiniPure donkey anti-rabbit IgG (H + L) (Jackson, #99212) for 1 h. After staining with DAPI for 1 h, the coverslips were mounted with antifade mounting medium (Beyotime, China, #P0126) on slides, and cells were observed with a confocal microscope (LSM 800, ZEISS, Germany).

NUNC Maxisorp Immunoplates were coated with 50 μL of 1 μg/mL ZGP42/3.7 at 4 °C overnight and then washed once with PBS containing 0.05% Tween 20 (PBST). The plate was blocked with 3% skim milk (190 μL/well) for 3 h at room temperature. After washing once with PBST, 50 μL of appropriately diluted serum or plasma samples or the concentrated sGPs in PBS containing 1% skim milk were added and incubated overnight at 4  °C. After washing 3 times with PBST, 50 μL of 1 μg/mL Rabbit anti-EBOV sGP pAb (IBT BIOSERVICES; 0365-001) was added and incubated for 1 h at room temperature. After washing 3 times with PBST, the bound antibodies were labeled by using 50 μL of 1:1000 peroxidase AffiniPure Donkey Anti-Rabbit IgG (H + L) (Jackson ImmunoResearch) diluted in 1% skim milk in PBST. After incubation for 1 h at room temperature and 3 PBST washes, 50 μL of KPL ABTS Peroxidase substrate solution mix (SeraCare Life Sciences) was added to each well, and the mixture was incubated for 15 min at room temperature. The optical density (OD) at 405 nm was measured. The standard curve for the sGP sandwich ELISA was carried out using serial dilutions of recombinant EBOV-sGP.

Tissues were fixed in 4% paraformaldehyde and embedded in paraffin, and serial 5-μm sections were prepared from the whole biopsy for immunohistochemistry. Sections were incubated for 1 h with a primary antibody to Collagen VI (Rabbit Polyclonal, ab6588, Abcam, Dilution 1:100) at RT in a humidity chamber, washed and incubated with fluorescence secondary antibody for 1 h [(Cy3-AffiniPure Donkey Anti-Rabbit IgG (H + L) (711-165-152, 1:200); Jackson Immunoresearch Laboratories] and DAPI as a counterstain. Tissue exposed to secondary antibody only was used as negative control.

Deparaffinized and rehydrated 3 μm paraffin sections of formaline-fixed kidneys were enzyme-treated by protease. The primary antibody used for this study was desmin (West Grove, PA, USA). Secondary antibodies used peroxidase-conjugated AffiniPure Donkey Anti-Rabbit IgG (H + L), purchased from Jackson ImmunoResearch (West Grove, PA, USA). Liquid DAB + Substrate Chromogen System (Dako, Tokyo, Japan) were used for the color reaction, after counterstaining with hematoxylin. For confirmation of the mesangial proliferative glomerulonephritis by inducing ethanol intake and restraint stress, the number of the mesangial cells was counted in the 65 glomerulus of the kidney.